Materials and methods for prevention and treatment of RNA viral diseases

ABSTRACT

The subject invention concerns a method of inhibiting an RNA virus infection within a patient by increasing the amount of 2-5 oligoadenylate synthetase (2-5 AS) activity within the patient. Preferably, the preventative and therapeutic methods of the present invention involve administering a nucleotide encoding 2-5 AS, or at least one catalytically active fragment thereof, such as the p40, p69, p100 subunits, to a patient in need thereof. The present inventors have determined that overexpression of 2-5AS causes a reduction in epithelial cell damage, reduction in infiltration of mononuclear cells in the peribronchiolar and perivascular regions, and reduction in thickening of the septa in the lungs. Levels of chemokines, such as MIP1-α, are also reduced upon overexpression of 2-5AS. The subject invention also pertains to pharmaceutical compositions containing a nucleotide sequence encoding 2-5 AS and a pharmaceutically acceptable carrier, as well as vectors for delivery of the 2-5 AS nucleotide sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.12/638,800, filed Dec. 15, 2009, which is a continuation of U.S.application Ser. No. 12/016,041, filed Jan. 17, 2008, which is adivisional of U.S. application Ser. No. 10/426,436, filed Apr. 30, 2003,now U.S. Pat. No. 7,354,908, which claims the benefit of provisionalpatent application Ser. No. 60/319,313, filed Jun. 12, 2002, andprovisional patent application Ser. No. 60/319,216, filed Apr. 30, 2002,all of which are hereby incorporated by reference in their entirety,including all nucleic acid sequences, amino acid sequences, figures,tables, and drawings.

BACKGROUND OF INVENTION

Respiratory syncytial virus (RSV) is a major respiratory pathogen ininfants, young children, and the elderly, causing severe bronchiolitis,pneumonia, and exacerbation of asthma. In the United States alone, RSVcauses approximately 4 million cases of respiratory tract infectionannually, which results in 149,000 hospitalizations and 11,000 deaths.It has been established that interferon-gamma (IFN-γ) gene therapy iseffective against RSV infection in BALB/c mice (Kumar et al., Vaccine18, 558-567, 1999).

Intranasal administration of a plasmid expressing IFN-γ cDNA proved tobe an effective prophylaxis in mice. Furthermore, IFN-γ expressed by arecombinant respiratory syncytial virus attenuates virus replication inmice without compromising immunogenicity. IFN-γ, a type II interferon,is a pleotropic cytokine which plays an important role in modulatingnearly all phases of immune and inflammatory responses. IFNs bind tospecific receptors on cells and activate the JAK-STAT signaling cascade,which culminates in the transcriptional induction of IFN-stimulatedgenes (ISGs). The Jak1 and Jak2 phosphorylate STAT-1 following thebinding of IFN-γ to its receptor. Once phosphorylated, STAT moleculesdimerize and translocate to the nucleus and bind to gamma activatedsequence (GAS) elements present in the regulatory regions of variousISGs. The antiviral mechanism of IFN-γ may involve one or more of anumber of ISG-encoded products, including interferon regulatory factor-1(IRF-1) double stranded RNA-dependent protein kinase (PKR), the Mxfamily of proteins, a family of 2′-5′-oligoadenylate synthetases (2-5AS), and RNase L.

RNase L is constitutively expressed in most mammalian cells and is foundin an inactive form bound to RNase L inhibitor (RLI), a 68 kDa proteinnot regulated by IFN-γ. The 2-5 AS produces a series of 5′phosphorylated, 2′, and 5′-linked oligoadenylates (2-5 A) from ATP, whenactivated by double-stranded ribonucleic acid (dsRNA). Upon binding of2-5 AS with RNase L, RLI is released and consequently, RNase L isdimerized and activated, mediating the cleavage of single-stranded RNA.However, the mechanism of the induction and activation of each of thesegenes is different in different cells and for the types of viruses. Themechanism of the IFN-γ-mediated anti-viral activity remains to beelucidated for many clinically important viruses.

BRIEF SUMMARY OF THE INVENTION

The present invention provides materials and methods useful forinhibiting viral infections caused by ribonucleic acid (RNA) virusesthat transiently produce double-stranded RNA during replication. Thesubject invention concerns therapeutic methods for preventing ordecreasing the severity of symptoms associated with an RNA viralinfection by increasing endogenous levels of 2′-5′ oligoadenylatesynthetase (2-5 AS) activity within the patient. Preferably, theendogenous levels of the 2-5 AS p40 subunit (e.g., the 40 kDa, 42 kDa,46 kDa, or other splice variants), p69 subunit, (e.g., the 69 kDa, 71kDa, or other splice variants), p100 subunit, or combinations thereof,are increased within the patient.

The materials and methods of present invention are effective fortreating or preventing human or animal infections from RNA viruses suchas, members of the family paramyxoviridae, respiratory syncytial virus(RSV), Rhinovirus, Vaccinia, Reovirus, human immunodeficiency virus(HIV), encephalomyocarditis virus (EMCV), Hepatitis B, Hepatitis C, aswell as bovine respiratory syncytial virus (BRSV), which infect cattle,sheep, and goats; Measles virus; Sendai virus; Parainfluenza 1, 2, and3; Mumps virus, Simian virus; and Newcastle virus.

In one aspect, the method of the present invention involves theadministration of a nucleotide sequence encoding 2-5 AS, or at least onecatalytically active fragment of 2-5 AS, such as the p40, p69, or p100subunits of 2-5 AS, to a patient in need thereof. The nucleotidesequence encoding 2-5 AS or at least one catalytically active fragmentthereof can be administered to the patient, for example, in a viralvector or non-viral vector, such as plasmid deoxyribonucleic acid (DNA).In cases wherein the RNA virus is one which infects the patient'srespiratory system, the nucleotide sequence encoding 2-5 AS, or at leastone catalytically active fragment thereof, is preferably administeredorally or intranasally to the epithelial mucosa of the respiratorysystem.

The present invention also pertains to pharmaceutical compositionscomprising a nucleotide sequence encoding 2-5 AS, or at least onecatalytically active fragment thereof, such as the p40, p69, or p100subunits of 2-5 AS, and a pharmaceutically acceptable carrier. Thepharmaceutical compositions of the present invention are useful forpreventing or decreasing the severity of symptoms associated with RNAviral infections. In another embodiment, the pharmaceutical compositionsof the present invention comprise the 2-5 AS polypeptide, or at leastone catalytically active fragment of the 2-5 AS polypeptide, and apharmaceutically acceptable carrier. The pharmaceutical compositions ofthe present invention can include various agents that protect thenucleic acid or amino acid contents from protein degradation.

In another aspect, the present invention concerns vectors containing anucleotide sequence encoding 2-5 AS, or at least one catalyticallyactive fragment thereof, such as the p40, p69, or p100 subunits of 2-5AS. Optionally, the vector can further include a promoter sequenceoperatively linked to the nucleotide encoding 2-5 AS or at least onecatalytically active fragment thereof, permitting expression of thenucleotide sequence within a host cell. In another aspect, the presentinvention includes host cells that have been genetically modified with anucleotide sequence encoding 2-5 AS, or at least one catalyticallyactive fragment thereof, such as the p40, p69, or p100 subunits of 2-5AS.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-D show the results of pre-incubation of HEp-2 cells for 4-20hours with different concentrations of IFN-γ and subsequent infectionwith RSV.

FIGS. 2A and 2B show results of a western blot analysis using specificantibodies to IRF-1, IRF-2, cytokeratin-18, double stranded RNA proteinkinase (PKR), and inducible nitric oxide synthase (iNOS). Proteins wereanalyzed from cells at various time points post treatment with IFN-γ(1000 U/ml). Cytokeratin-18 was used as an internal control.

FIG. 3 show results of northern analysis performed using gene specificprobes for IRF-1 and the p40 and p69 isoforms of 2-5 AS.

FIG. 4 shows results of exposure of HEp-2 cells to IFN-γ (1000 U/ml at20 hours pre-infection) and treatment with equimolar mixtures ofantisense oligonucleotides (ODNs) to both p40 and p69 isoforms of 2-5AS. Scrambled mismatch of the antisense ODN sequence to p40 and p69 atthe same concentration were used as control.

FIGS. 5A-D show the results of northern analyses of (i) RNAs from RNAseL inhibitor (RLI) and HEp-2 using a gene specific probe for RLI and (ii)the level of mRNA expression of IRF-1, p40, and p69 isoforms of 2-5 AS.

FIG. 6 show the results of treatment of both HEp-2 cells and RLI-14cells with IFN-γ (at 100-1000 U/ml at 20 hours pre-infection) andsubsequent infection with RSV.

FIG. 7 shows the results of treatment of HEp-2 cells with IFN-γ (at100-1000 U/ml at 20 hours pre-infection) and subsequent infection withRSV. 2-5 A was transfected at 3 hours prior to RSV infection. Cells wereharvested at 72 hours post infection and the clear cell homogenate wasused for the RSV plaque assay (***: p<0.005; ††: p<0.05).

FIGS. 8A-8C show lung titers of RSV in infected mice following 2-5 AScDNA vaccination. BALB/c mice (n=4) were intranasally administered withp2′-5′ AS (25 mg of DNA each time complexed with lipofectamine) or anequal amount of empty pVAX (CLONTECH, Palo Alto, Calif., USA) vector DNA(as a transfection control) 3 times in 2 day intervals. As shown in FIG.8A, 2-5 AS cDNA vaccination significantly attenuated lung titers of RSV.FIG. 8B shows that vaccination with 2-5 AS cDNA decreases production ofthe chemokine macrophage inflammatory protein-1 alpha (MIP-1 α). Theresults of bronchoalveolar lavage (BAL) cell differential (FIG. 8C) showthat 2-5 AS does not significantly alter the cellular composition of thelung, although the percent of neutrophils is increased in the lungs ofmice treated with 2-5 AS cDNA.

FIGS. 9A-9C show representative photomicrographs of lungs stained withhematoxylin and eosin (H & E). FIG. 9A is an untreated control. FIGS. 9Band 9C show histological sections of RSV infected lungs followingtreatment with the empty pVAX vector and p2′-5′ AS, respectively.

FIG. 10 shows results of treatment with adenoviral vector (Ad)-2-5 AS(p40) results in attenuation of RSV replication. BALB/c mice wereintranasally administered with Ad-p40 and then infected with RSV. Lungswere harvested five days post RSV infection and RSV replication wasassayed by RT-PCR analysis of RSV-N gene. GAPDH was used as internalcontrol.

FIG. 11 shows that Ad-p40 attenuates RSV lung titers. Mice wereintranasally given Ad-p40 and then infected with RSV. Lungs wereharvested five days post RSV infection and lung homogenate was used forRSV plaque assay. Ad-LacZ was used as control.

FIG. 12 shows that Ad-p40 inhibits RSV induced airway reactivity. BALB/cmice were intranasally administered with Ad-p40 and subsequentlyinfected with RSV. AHR was measured on day 4 post-RSV infection. Ad-p40treatment significantly decreased pulmonary inflammation.

FIGS. 13A-13H show that Ad-p40 overexpression normalizes macrophage andlymphocyte numbers in the lung in RSV infected mice. BAL celldifferential was performed and percentages of macrophage, lymphocytesand neutrophils was determined. Both Ad-IFNg and Ad-p40 treatmentreduced the lymphocyte population to normal, compared to RSV-infectedmice. Histological sections from lungs were stained with hematoxylin andeosin and representative photomicrographs are shown. Sections shown areas follows: Naive mice (FIG. 13A; with magnified inset FIG. 13B); RSVinfected mice (FIG. 13C; with magnified inset, FIG. 13D); Ad-p40 treatedmice (FIG. 13E; with magnified inset, FIG. 13F); and Ad-lacZ treatedmice (FIG. 13G; with magnified inset, FIG. 13H).

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 is a nucleotide coding sequence (CDS) for the human 40 kDasplice variant of the 40/46 kDa subunit (“p40 subunit”) of 2′-5′oligoadenylate synthetase (National Center for Biotechnology Information(NCBI) Accession Number NM_(—)016816).

SEQ ID NO: 2 is an amino acid sequence of the human 40 kDa splicevariant of the 40/46 kDa subunit (“p40 subunit”) of 2′-5′ oligoadenylatesynthetase (NCBI Accession Number NM_(—)016816).

SEQ ID NO: 3 is a nucleotide coding sequence (CDS) for the human 46 kDAsplice variant of the 40/46 kDa subunit (“p40 subunit”) of 2′-5′oligoadenylate synthetase (National Center for Biotechnology Information(NCBI) Accession Number NM_(—)016816).

SEQ ID NO: 4 is an amino acid sequence of the human 46 kDA splicevariant of the 40/46 kDa subunit (“p40 subunit”) of 2′-5′ oligoadenylatesynthetase (NCBI Accession Number NM_(—)016816).

SEQ ID NO: 5 is a nucleotide coding sequence (CDS) for the human 69 kDAsplice variant of the 69/71 kDa subunit (“p69 subunit”) of 2′-5′oligoadenylate synthetase (NCBI Accession Number NM_(—)002535).

SEQ ID NO: 6 is an amino acid sequence of the human 69 kDa splicevariant of the 69/71 kDa subunit (“p69 subunit”) of 2′-5′ oligoadenylatesynthetase (NCBI Accession Number NM_(—)002535).

SEQ ID NO: 7 is a nucleotide coding sequence (CDS) for the human 71 kDAsplice variant of the 69/71 kDa subunit (“p69 subunit”) of 2′-5′oligoadenylate synthetase (NCBI Accession Number NM_(—)002535).

SEQ ID NO: 8 is an amino acid sequence of the human 71 kDa splicevariant of the 69/71 kDa subunit (“p69 subunit”) of 2′-5′ oligoadenylatesynthetase (NCBI Accession Number NM_(—)002535).

SEQ ID NO: 9 is a nucleotide coding sequence (CDS) for the human 100 kDasubunit (“p100 subunit”) of 2′-5′ oligoadenylate synthetase (NCBIAccession Number AF063613).

SEQ ID NO: 10 is an amino acid sequence of the human 100 kDa subunit(“p100 subunit”) of 2′-5′ oligoadenylate synthetase (NCBI AccessionNumber AF063613).

SEQ ID NO: 11 is a nucleotide coding sequence (CDS) for the mousehomolog of the 2′-5′ oligoadenylate synthetase 40 kDa splice variant(p40 subunit) (NCBI Accession Number M33863).

SEQ ID NO: 12 is the amino acid sequence for the mouse homolog of the2′-5′ oligoadenylate synthetase 40 kDa splice variant (p40 subunit)(NCBI Accession Number M33863).

SEQ ID NO: 13 is the human 2′-5′ oligoadenylate synthetase 40/46 kDa(p40 subunit) gene (NCBI Accession Number NM_(—)016816).

SEQ ID NO: 14 is the human 2′-5′ oligoadenylate synthetase 69/71 kDa(p69 subunit) gene (NCBI Accession Number NM_(—)002535).

SEQ ID NO: 15 is the human 2′-5′ oligoadenylate synthetase 100 kDa (p100subunit) gene (NCBI Accession Number AF063613).

SEQ ID NO: 16 is the mouse homolog of the 2′-5′ oligoadenylatesynthetase 40 kDa (p40 subunit) gene (NCBI Accession Number M33863).

SEQ ID NO: 17 is a phosphorothioate antisense oligonucleotide (ODN)designed against the p40 subunit of 2′-5′ oligoadenylate synthetase.

SEQ ID NO: 18 is an ODN designed against the p69 subunit of 2′-5′oligoadenylate synthetase.

SEQ ID NO: 19 is a scramble of the antisense sequence to p40, i.e.,identical in base composition.

SEQ ID NO: 20 is a scramble of the antisense sequence to p69.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns a method of inhibiting an RNA virusinfection within a patient by increasing the endogenous levels of 2-5oligoadenylate synthetase (2-5 AS) activity within the patient.Preferably, the endogenous levels of the 2-5 AS p40 subunit (e.g., 40kDa, 42 kDA, 46 kDa, or other splice variant), p69 subunit (e.g., 69kDa, 71 kDa, or other splice variant), p100 subunit, or combinationsthereof, are increased within the patient.

The present inventors have determined that overexpression of the 2-5 AS,or catalytically active fragments thereof, causes a reduction inepithelial cell damage, reduction in infiltration of mononuclear cellsin the peribronchiolar and perivascular regions, and reduction in thethickening of the septa in the lungs of patients suffering fromrespiratory RNA viruses, such as respiratory syncytial virus (RSV).Levels of chemokines, such as MIP1-α, are also reduced uponoverexpression of 2-5 AS.

Infections from members of the family paramyxoviridae that producedouble-stranded RNA as a requirement of replication can be prevented ortreated using the present invention. Thus, infections by members of thegenera paramyxovirus, morbillivirus, rubulavirus, pnuemovirus, andothers can be inhibited in humans and animals. Examples of RNA virusesthat produce double-stranded RNA during intermediate replication andwhich infect humans include, but are not limited to, respiratorysyncytial virus (RSV), Rhinovirus, Vaccinia, Reovirus, HIV, EMCV,Hepatitis B, and Hepatitis C. Examples of RNA viruses that infectanimals and produce double-stranded RNA during intermediate replicationinclude, but are not limited to, bovine respiratory syncytial virus(BRSV), which infect cattle, sheep, and goats; Measles virus; Sendaivirus; Parainfluenza 1, 2, and 3; Mumps virus, Simian virus; andNewcastle virus. Infections caused by coronavirus (such as thatresponsible for severe acute respiratory syndrome (SARS)), rotavirus,parainfluenza virus, West Nile virus, as well as other viruses in whichinterferon actively inhibits viral replication can be inhibited usingthe methods and compositions of the present invention.

In one aspect, the subject invention concerns a method of treating orpreventing an RNA virus infection within a patient by increasing the invivo concentration of 2-5 AS or a catalytically active fragment thereofwithin the patient, thereby inhibiting the RNA virus infection.Preferably, the methods of the present invention do not involveadministration of interferon or a polynucleotide encoding interferon,such as interferon-alpha (IFN-α), interferon-beta (IFN-β), orinterferon-gamma (IFN-γ), or the administration of such IFNpolypeptides. Thus, the methods and compositions of the presentinvention are directed to increasing the in vivo concentration of 2-5 ASor a catalytically active fragment thereof, which is an IFN-γ-induceddownstream molecule. Advantageously, the methods and compositions of thepresent invention exhibit an antiviral effect without the adverseeffects associated with IFN-γ.

The in vivo concentration of the 2-5 AS, or a catalytically activefragment thereof, can be increased, for example, by exogenousadministration of the 2-5 AS polypeptide, or a catalytically activefragment of the polypeptide. Preferably, the in vivo concentration ofthe 2-5 AS polypeptide or catalytically active fragment is increased byincreasing or up-regulating the functional expression of the nucleotidesequence encoding 2-5 AS or at least one catalytically active fragmentthereof, such as the p40, p69, or p100 subunits, as gene therapy. Morepreferably, a nucleotide sequence encoding 2-5 AS or at least onecatalytically active fragment thereof can be administered to a patientand expressed in order to increase the endogenous level of 2-5 ASenzymatic activity within the patient. For example, at least onenucleotide sequence selected from the group consisting of SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 14, 15, 16, and 17, or a catalytically activefragment thereof, can be administered to the patient. The nucleotidesequence can be administered to a patient's cells in vivo or in vitro(including ex vivo, genetically modifying the patient's own cells exvivo and subsequently administering the modified cells back into thepatient).

In another aspect of the invention, 2-5 AS polypeptide, or at least onecatalytically active fragment thereof, is administered to a patient inorder to increase the antiviral function of 2-5 AS within the patient.Preferably, the polypeptides utilized are those disclosed herein. Thepolypeptides can comprise catalytically active fragments of thefull-length 2-5 AS amino acid sequence, such as the p40, p69, or p100subunits, including splice variants of these subunits, or mammalianhomologs of these subunits (e.g., the p46 isoform of OAS-1; accessionnumber NP_(—)058132.1), such as murine homologs. For example, thepolypeptides can comprise one or more amino acid sequences set forthherein as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 14, 15 or 16, orcatalytically active fragments of these amino acid sequences.

Various means for delivering polypeptides to a cell can be utilized tocarry out the methods of the subject invention. For example, proteintransduction domains (PTDs) can be fused to the polypeptide, producing afusion polypeptide, in which the PTDs are capable of transducing thepolypeptide cargo across the plasma membrane (Wadia, J. S, and Dowdy, S.F., Curr. Opin. Biotechnol., 2002, 13 (1)52-56). Examples of PTDsinclude the Drosophila homeotic transcription protein antennapedia(Antp), the herpes simplex virus structural protein VP22, and the humanimmuno-deficiency virus 1 (HIV-1) transcriptional activator Tat protein.

According to the method of RNA virus inhibition of the subjectinvention, recombinant cells can be administered to a patient, whereinthe recombinant cells have been genetically modified to express the geneencoding 2-5 AS or at least one catalytically active fragment thereof,such as the p40, p69, or p100 subunits of 2-5 AS. If the cells to begenetically modified already express a gene encoding 2-5 AS, the geneticmodification can serve to enhance or increase expression of the geneencoding 2-5 AS or a catalytically active fragment of 2-5 AS beyond thenormal or constitutive amount (e.g., “overexpression”).

The method of RNA virus inhibition of the subject invention can be usedto treat a patient suffering from an RNA virus infection, or as apreventative of RNA virus infection (i.e., prophylactic treatment).According to the methods of the subject invention, various othercompounds, such as antiviral compounds, can be administered inconjunction with (before, during, or after) increasing the in vivoconcentrations of 2-5 AS or at least one catalytically active fragmentwithin the patient. Various compositions and methods for preventing ortreating RNA virus infection can be used in conjunction with thecompositions and methods of the subject invention, such as thosedescribed in U.S. Pat. No. 6,489,306, filed Feb. 23, 1999, and U.S.published patent application Serial No. 2003/00068333, filed Feb. 12,2002, which are incorporated herein by reference in their entirety. Forexample, nucleotide sequences encoding 2-5 AS or at least onecatalytically active fragment thereof can be conjugated with chitosan, abiodegradable, human-friendly cationic polymer that increases mucosalabsorption of the gene expression vaccine without any adverse effects,as described in U.S. published patent application Serial No.2003/00068333.

The nucleotide sequence can be formulated in the form of nanosphereswith chitosan. Chitosan allows increased bioavailability of the DNAbecause of protection from degradation by serum nucleases in the matrixand thus has great potential as a mucosal gene delivery system, forexample. Chitosan exhibits various beneficial effects, such asanticoagulant activity, wound-healing properties, and immunostimulatoryactivity, and is capable of modulating immunity of the mucosa andbronchus-associated lymphoid tissue.

Nucleotide, polynucleotide, or nucleic acid sequences(s) are understoodto mean, according to the present invention, either a double-strandedDNA, a single-stranded DNA, products of transcription of the said DNAs(e.g., RNA molecules), or corresponding RNA molecules that are notproducts of transcription. It should also be understood that the presentinvention does not relate to the genomic nucleotide sequences encoding2-5 AS or catalytically active fragments thereof in their natural/nativeenvironment or natural/native state. The nucleic acid, polynucleotide,or nucleotide sequences of the invention have been isolated, purified(or partially purified), by separation methods including, but notlimited to, ion-exchange chromatography, molecular size exclusionchromatography, affinity chromatography, or by genetic engineeringmethods such as amplification, cloning or subcloning.

Optionally, the polynucleotide sequence encoding 2-5 AS or catalyticallyactive fragment thereof can also contain one or more polynucleotidesencoding heterologous polypeptide sequences (e.g., tags that facilitatepurification of the polypeptides of the invention (see, for example,U.S. Pat. No. 6,342,362, hereby incorporated by reference in itsentirety; Altendorf et al. [1999-WWW, 2000] “Structure and Function ofthe F_(o) Complex of the ATP Synthase from Escherichia Coli,” J. ofExperimental Biology 203:19-28, The Co. of Biologists, Ltd., G. B.;Baneyx [1999] “Recombinant Protein Expression in Escherichia coli,”Biotechnology 10:411-21, Elsevier Science Ltd.; Eihauer et al. [2001]“The FLAG Peptide, a Versatile Fusion Tag for the Purification ofRecombinant Proteins,” J. Biochem Biophys Methods 49:455-65; Jones etal. [1995] J. Chromatography 707:3-22; Jones et al. [1995] “CurrentTrends in Molecular Recognition and Bioseparation,” J. of ChromatographyA. 707:3-22, Elsevier Science B. V.; Margolin [2000] “Green FluorescentProtein as a Reporter for Macromolecular Localization in BacterialCells,” Methods 20:62-72, Academic Press; Puig et al. [2001] “The TandemAffinity Purification (TAP) Method: A General Procedure of ProteinComplex Purification,” Methods 24:218-29, Academic Press; Sassenfeld[1990] “Engineering Proteins for Purification,” TibTech 8:88-93;Sheibani [1999]“Prokaryotic Gene Fusion Expression Systems and Their Usein Structural and Functional Studies of Proteins,” Prep. Biochem. &Biotechnol. 29(1):77-90, Marcel Dekker, Inc.; Skerra et al. [1999]“Applications of a Peptide Ligand for Streptavidin: the Strep-tag”,Biomolecular Engineering 16:79-86, Elsevier Science, B. V.; Smith [1998]“Cookbook for Eukaryotic Protein Expression: Yeast, Insect, and PlantExpression Systems,” The Scientist 12 (22):20; Smyth et al. [2000]“Eukaryotic Expression and Purification of Recombinant ExtracellularMatrix Proteins Carrying the Strep II Tag”, Methods in MolecularBiology, 139:49-57; Unger [1997] “Show Me the Money: ProkaryoticExpression Vectors and Purification Systems,” The Scientist 11 (17):20,each of which is hereby incorporated by reference in their entireties),or commercially available tags from vendors such as such as STRATAGENE(La Jolla, Calif.), NOVAGEN (Madison, Wis.), QIAGEN, Inc., (Valencia,Calif.), or INVITROGEN (San Diego, Calif.).

Other aspects of the invention provide vectors containing one or more ofthe polynucleotides of the invention, such as vectors containingnucleotides encoding 2-5 AS or catalytically active fragments of 2-5 AS,such as the p40 and/or p69 subunits. The vectors can be vaccine,replication, or amplification vectors. In some embodiments of thisaspect of the invention, the polynucleotides are operably associatedwith regulatory elements capable of causing the expression of thepolynucleotide sequences. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, lentiviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations of the aforementioned vector sources, such as those derivedfrom plasmid and bacteriophage genetic elements (e.g., cosmids andphagemids). Preferably, the vector is an adenoaviral vector oradeno-associated virus vector.

As indicated above, vectors of this invention can also comprise elementsnecessary to provide for the expression and/or the secretion of 2-5 AS,or a catalytically active fragment thereof, encoded by the nucleotidesequences of the invention in a given host cell. The vector can containone or more elements selected from the group consisting of a promotersequence, signals for initiation of translation, signals for terminationof translation, and appropriate regions for regulation of transcription.In certain embodiments, the vectors can be stably maintained in the hostcell and can, optionally, contain signal sequences directing thesecretion of translated protein. Other embodiments provide vectors thatare not stable in transformed host cells. Vectors can integrate into thehost genome or be autonomously-replicating vectors.

In a specific embodiment, a vector comprises a promoter operably linkedto a 2-5 AS-encoding nucleic acid sequence (or a catalytically activefragment thereof), one or more origins of replication, and, optionally,one or more selectable markers (e.g., an antibiotic resistance gene).Non-limiting exemplary vectors for the expression of the polypeptides ofthe invention include pBr-type vectors, pET-type plasmid vectors(PROMEGA), pBAD plasmid vectors (INVITROGEN), and pVAX plasmid vectors(INVITROGEN), or others provided in the examples below. Furthermore,vectors according to the invention are useful for transforming hostcells for the cloning or expression of the nucleotide sequences of theinvention.

Promoters which may be used to control expression include, but are notlimited to, the CMV promoter, the SV40 early promoter region (Bernoistand Chambon [1981] Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al. [1980] Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al. [1981]Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al. [1982] Nature 296:39-42);prokaryotic vectors containing promoters such as the β-lactamasepromoter (Villa-Kamaroff, et al. [1978] Proc. Natl. Acad. Sci. USA75:3727-3731), or the tac promoter (DeBoer, et al. [1983] Proc. Natl.Acad. Sci. USA 80:21-25); the lung specific promoters such as surfactantprotein B promoter (Venkatesh et al., Am. J. Physiol. 268 (Lung CellMol. Physiol. 12):L674-L682, 1995); see also, “Useful Proteins fromRecombinant Bacteria” in Scientific American, 1980, 242:74-94; plantexpression vectors comprising the nopaline synthetase promoter region(Herrera-Estrella et al. [1983] Nature 303:209-213) or the cauliflowermosaic virus 35S RNA promoter (Gardner, et al. [1981] Nucl. Acids Res.9:2871), and the promoter of the photosynthetic enzyme ribulosebiphosphate carboxylase (Herrera-Estrella et al. [1984] Nature310:115-120); promoter elements from yeast or fungi such as the Gal 4promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerolkinase) promoter, and/or the alkaline phosphatase promoter.

The subject invention also provides for “homologous” or “modified”nucleotide sequences. Modified nucleic acid sequences will be understoodto mean any nucleotide sequence obtained by mutagenesis according totechniques well known to persons skilled in the art, and exhibitingmodifications in relation to the normal sequences. For example,mutations in the regulatory and/or promoter sequences for the expressionof a polypeptide that result in a modification of the level ofexpression of a polypeptide according to the invention provide for a“modified nucleotide sequence”. Likewise, substitutions, deletions, oradditions of nucleic acid to the polynucleotides of the inventionprovide for “homologous” or “modified” nucleotide sequences. In variousembodiments, “homologous” or “modified” nucleic acid sequences havesubstantially the same biological activity as the native (naturallyoccurring) 2-5 AS or subunit thereof. A “homologous” or “modified”nucleotide sequence will also be understood to mean a subunit or asplice variant of the polynucleotides of the instant invention or anynucleotide sequence encoding a “modified polypeptide” as defined below.

A homologous nucleotide sequence, for the purposes of the presentinvention, encompasses a nucleotide sequence having a percentageidentity with the bases of the nucleotide sequences of between at least(or at least about) 20.00% to 99.99% (inclusive), and which encodes acatalytically active polypeptide. The aforementioned range of percentidentity is to be taken as including, and providing written descriptionand support for, any fractional percentage, in intervals of 0.01%,between 20.00% and 99.99%. These percentages are purely statistical anddifferences between two nucleic acid sequences can be distributedrandomly and over the entire sequence length.

In various embodiments, homologous sequences exhibiting a percentageidentity with the bases of the nucleotide sequences of the presentinvention can have 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identitywith the polynucleotide sequences of the instant invention.

Both protein and nucleic acid sequence homologies may be evaluated usingany of the variety of sequence comparison algorithms and programs knownin the art. Such algorithms and programs include, but are by no meanslimited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson andLipman [1988] Proc. Natl. Acad. Sci. USA 85 (8):2444-2448; Altschul etal. [1990] J. Mol. Biol. 215 (3):403-410; Thompson et al. [1994] NucleicAcids Res. 22 (2):4673-4680; Higgins et al. [1996] Methods Enzymol.266:383-402; Altschul et al. [1990] J. Mol. Biol. 215 (3):403-410;Altschul et al. [1993] Nature Genetics 3:266-272).

Nucleotide sequences encoding polypeptides with enhanced 2-5 AScatalytic activity can be obtained by “gene shuffling” (also referred toas “directed evolution”, and “directed mutagenesis”), and used in thecompositions and methods of the present invention. Gene shuffling is aprocess of randomly recombining different sequences of functional genes(recombining favorable mutations in a random fashion) (U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; and 5,837,458). Thus, proteinengineering can be accomplished by gene shuffling, random complexpermutation sampling, or by rational design based on three-dimensionalstructure and classical protein chemistry (Cramer et al., Nature,391:288-291, 1998; and Wulff et al., The Plant Cell, 13:255-272, 2001).

The subject invention also provides nucleotide sequences complementaryto any of the polynucleotide sequences disclosed herein. Thus, theinvention is understood to include any DNA whose nucleotides arecomplementary to those of 2-5 AS polynucleotide sequence of theinvention, or catalytically active fragments thereof, and whoseorientation is reversed (e.g., an antisense sequence).

The present invention further provides catalytically active fragments ofthe 2-5 AS polynucleotide sequences, including catalytically activefragments of the 2-5 AS subunit nucleotide sequences, provided herein.Representative fragments of the polynucleotide sequences according tothe invention will be understood to mean any nucleotide fragment havingat least 8 or 9 successive nucleotides, preferably at least 12successive nucleotides, and still more preferably at least 15 or atleast 20 successive nucleotides of the sequence from which it isderived. The upper limit for such fragments is the total number ofpolynucleotides found in the full-length sequence (or, in certainembodiments, of the full length open reading frame (ORF) identifiedherein). It is understood that, optionally, such fragments refer only toportions of the disclosed polynucleotide sequences that are not listedin a publicly available database or prior art references. However, itshould be understood that with respect to the method for inhibiting RSVof the subject invention, disclosed nucleotides (and polypeptidesencoded by such nucleotides) that are listed in a publicly availabledatabase or prior art reference can also be utilized. For example,nucleotide sequences that are 2-5 AS p40 or p69 subunit homologs, orfragments thereof, which have been previously identified, can beutilized to carry out the method for inhibiting RNA virus infection ofthe subject invention.

In other embodiments, fragments contain from one nucleotide less thanthe full length 2-5 AS enzyme, or from one nucleotide less than acatalytically active subunit thereof, such as p40 or p69 subunitpolynucleotide CDS sequences (e.g., 1,203 and 1,207 nucleotides for the40 kDa splice variant and 46 kDa splice variant, respectively; and 2063and 2,168 nucleotides for the 69 kDa splice variant and 71 kDa splicevariant, respectively) to fragments containing the smallest number ofnucleotides encoding a polypeptide that retains at least some 2-5 ASenzymatic activity.

Among these representative fragments, those capable of hybridizing understringent conditions with a nucleotide sequence encoding 2-5 AS orsubunits thereof are preferred. Conditions of high or intermediatestringency are provided infra and are chosen to allow for hybridizationbetween two complementary DNA fragments. Hybridization conditions for apolynucleotide of about 1,000 to 3,000 bases in size will be adapted bypersons skilled in the art for larger- or smaller-sizedoligonucleotides, according to methods well known in the art (see, forexample, Sambrook et al. [1989] Molecular Cloning, A Laboratory Manual,Second Edition, Cold Spring Harbor Press, N.Y., pp. 9.47-9.57).

The subject invention also provides detection probes (e.g., fragments ofthe disclosed polynucleotide sequences) for hybridization with a targetsequence or an amplicon generated from the target sequence. Such adetection probe will advantageously have as sequence a sequence of atleast 9, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, or 100 nucleotides. Alternatively, detection probes can comprise8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127 and up to, for example, 1,203 consecutive nucleotides, 1,207consecutive nucleotides, 2,064 consecutive nucleotides, 2,186consecutive nucleotides, 3,264 consecutive nucleotides, and 1,104consecutive nucleotides of those disclosed herein, which correspond,respectively, to the human 40 kDa splice variant of the 2-5 AS p40subunit (SEQ ID NO:1), human 46 kDa splice variant 2-5 AS p40 subunit(SEQ ID NO:3), human 69 kDa splice variant of the 2-5 AS p69 subunit(SEQ ID NO:5), human 71 kDa splice variant of the 2-5 AS p69 subunit(SEQ ID NO:7), human p100 subunit (SEQ ID NO:9), and the mouse homologof the 2-5 AS p40 subunit (SEQ ID NO: 11). The detection probes can alsobe used as labeled probe or primer in the subject invention. Labeledprobes or primers are labeled with a radioactive compound or withanother type of label. Alternatively, non-labeled nucleotide sequencesmay be used directly as probes or primers; however, the sequences aregenerally labeled with a radioactive element (³²P, ³⁵S, ³H, ¹²⁵I) orwith a molecule such as biotin, acetylaminofluorene, digoxigenin,5-bromo-deoxyuridine, or fluorescein to provide probes that can be usedin numerous applications.

The nucleotide sequences according to the invention may also be used inanalytical systems, such as DNA chips. DNA chips and their uses are wellknown in the art and (see for example, U.S. Pat. Nos. 5,561,071;5,753,439; 6,214,545; Schena et al. [1996] BioEssays 18:427-431; Bianchiet al. [1997] Clin. Diagn. Virol. 8:199-208; each of which is herebyincorporated by reference in their entireties) and/or are provided bycommercial vendors such as AFFYMETRIX, Inc. (Santa Clara, Calif.).

Various degrees of stringency of hybridization can be employed. The moresevere the conditions, the greater the complementarity that is requiredfor duplex formation. Severity of conditions can be controlled bytemperature, probe concentration, probe length, ionic strength, time,and the like. Preferably, hybridization is conducted under moderate tohigh stringency conditions by techniques well known in the art, asdescribed, for example, in Keller, G. H., M. M. Manak [1987] DNA Probes,Stockton Press, New York, N.Y., pp. 169-170.

By way of example, hybridization of immobilized DNA on Southern blotswith ³²P-labeled gene-specific probes can be performed by standardmethods (Maniatis et al. [1982] Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, New York). In general, hybridization andsubsequent washes can be carried out under moderate to high stringencyconditions that allow for detection of target sequences with homology tothe exemplified polynucleotide sequence. For double-stranded DNA geneprobes, hybridization can be carried out overnight at 20-25° C. belowthe melting temperature (Tm) of the DNA hybrid in 6×SSPE, 5×Denhardt'ssolution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature isdescribed by the following formula (Beltz et al. [1983] Methods ofEnzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, NewYork 100:266-285).T _(m)=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.61(% formamide)−600/lengthof duplex in base pairs.

Washes are typically carried out as follows:

-   -   (1) twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS        (low stringency wash);    -   (2) once at T_(m)-20EC for 15 minutes in 0.2×SSPE, 0.1% SDS        (moderate stringency wash).

For oligonucleotide probes, hybridization can be carried out overnightat 10-20° C. below the melting temperature (T_(m)) of the hybrid in6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. T_(m)for oligonucleotide probes can be determined by the following formula:T _(m)(° C.)=2(number T/A base pairs)+4(number G/C base pairs) (Suggs etal. [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D. D. Brown[ed.], Academic Press, New York, 23:683-693).

Washes can be carried out as follows:

-   -   (1) twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS        (low stringency wash;    -   2) once at the hybridization temperature for 15 minutes in        1×SSPE, 0.1% SDS (moderate stringency wash).

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment>70 or so bases in length, the followingconditions can be used:

-   -   Low: 1 or 2×SSPE, room temperature    -   Low: 1 or 2×SSPE, 42° C.    -   Moderate: 0.2× or 1×SSPE, 65° C.    -   High: 0.1×SSPE, 65° C.        By way of another non-limiting example, procedures using        conditions of high stringency can also be performed as follows:        Pre-hybridization of filters containing DNA is carried out for 8        h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM        Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02%        BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are        hybridized for 48 h at 65° C., the preferred hybridization        temperature, in pre-hybridization mixture containing 100 g/ml        denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled        probe. Alternatively, the hybridization step can be performed at        65° C. in the presence of SSC buffer, 1×SSC corresponding to        0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes        can be done at 37° C. for 1 h in a solution containing 2×SSC,        0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in        0.1×SSC at 50° C. for 45 min. Alternatively, filter washes can        be performed in a solution containing 2×SSC and 0.1% SDS, or        0.5×SSC and 0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15        minute intervals. Following the wash steps, the hybridized        probes are detectable by autoradiography. Other conditions of        high stringency which may be used are well known in the art        (see, for example, Sambrook et al. [1989] Molecular Cloning, A        Laboratory Manual, Second Edition, Cold Spring Harbor Press,        N.Y., pp. 9.47-9.57; and Ausubel et al. [1989] Current Protocols        in Molecular Biology, Green Publishing Associates and Wiley        Interscience, N.Y., each incorporated herein in its entirety).

A further non-limiting example of procedures using conditions ofintermediate stringency are as follows: Filters containing DNA arepre-hybridized, and then hybridized at a temperature of 60° C. in thepresence of a 5×SSC buffer and labeled probe. Subsequently, filterwashes are performed in a solution containing 2×SSC at 50° C. and thehybridized probes are detectable by autoradiography. Other conditions ofintermediate stringency which may be used are well known in the art(see, for example, Sambrook et al. [1989] Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp.9.47-9.57; and Ausubel et al. [1989] Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N.Y., eachof which is incorporated herein in its entirety).

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probe sequences ofthe subject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions and deletions can be produced in a givenpolynucleotide sequence in many ways, and these methods are known to anordinarily skilled artisan. Other methods may become known in thefuture.

It is also well known in the art that restriction enzymes can be used toobtain functional fragments of the subject DNA sequences. For example,Bal31 exonuclease can be conveniently used for time-controlled limiteddigestion of DNA (commonly referred to as “erase-a-base” procedures).See, for example, Maniatis et al. [1982] Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York; Wei et al. [1983] J.Biol. Chem. 258:13006-13512. The nucleic acid sequences of the subjectinvention can also be used as molecular weight markers in nucleic acidanalysis procedures.

The invention also provides host cells transformed by a polynucleotideaccording to the invention and the production of 2-5 AS or acatalytically active fragment thereof, by the transformed host cells. Insome embodiments, transformed cells comprise an expression vectorcontaining 2-5 AS nucleotide sequences or a catalytically activefragment thereof. Other embodiments provide for host cells transformedwith nucleic acids. Yet other embodiments provide transformed cellscomprising an expression vector containing fragments of 2-5 AS p40and/or p69 subunit nucleotide sequences. Transformed host cellsaccording to the invention are cultured under conditions allowing thereplication and/or the expression of the 2-5 AS nucleotide sequence or acatalytically active fragment thereof, such as the p40 and/or p69subunits. Expressed polypeptides are recovered from culture media andpurified, for further use, according to methods known in the art.

The host cell may be chosen from eukaryotic or prokaryotic systems, forexample bacterial cells (Gram negative or Gram positive), yeast cells,animal cells, human cells, plant cells, and/or insect cells usingbaculovirus vectors. In some embodiments, the host cell for expressionof the polypeptides include, and are not limited to, those taught inU.S. Pat. Nos. 6,319,691; 6,277,375; 5,643,570; 5,565,335; Unger [1997]The Scientist 11 (17):20; or Smith [1998] The Scientist 12(22):20, eachof which is incorporated by reference in its entirety, including allreferences cited within each respective patent or reference. Otherexemplary, and non-limiting, host cells include Staphylococcus spp.,Enterococcus spp., E. coli, and Bacillus subtilis; fungal cells, such asStreptomyces spp., Aspergillus spp., S. cerevisiae, Schizosaccharomycespombe, Pichia pastoris, Hansela polymorpha, Kluveromyces lactis, andYarrowia lipolytica; insect cells such as Drosophila S2 and SpodopteraSf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells. A great variety of expressionsystems can be used to produce the 2-5 AS polypeptides or catalyticallyactive fragments thereof and encoding polynucleotides can be modifiedaccording to methods known in the art to provide optimal codon usage forexpression in a particular expression system.

Furthermore, a host cell strain may be chosen that modulates theexpression of the inserted sequences, modifies the gene product, and/orprocesses the gene product in the specific fashion. Expression fromcertain promoters can be elevated in the presence of certain inducers;thus, expression of the genetically engineered polypeptide may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation) ofproteins. Appropriate cell lines or host systems can be chosen to ensurethe desired modification and processing of the foreign proteinexpressed. For example, expression in a bacterial system can be used toproduce an unglycosylated core protein product whereas expression inyeast will produce a glycosylated product. Expression in mammalian cellscan be used to provide “native” glycosylation of a heterologous protein.Furthermore, different vector/host expression systems may effectprocessing reactions to different extents.

Nucleic acids and/or vectors encoding 2-5 AS, or catalytically activefragments thereof, such as the p40 and/or p69 subunits, can beintroduced into host cells by well-known methods, such as, calciumphosphate transfection, DEAE-dextran mediated transfection,transfection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection (see, for example, Sambrook et al. [1989] MolecularCloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

The subject invention also provides for the expression of the 2-5 AS p40or p69 subunit, derivative, or an analogue (e.g., a splice variant)encoded by a polynucleotide sequence disclosed herein. Alternatively,the invention provides for the expression of a polynucleotide encoding afragment of a 2-5 AS p40 or p69 subunit. In either embodiment, thedisclosed sequences can be regulated by a second nucleic acid sequenceso that the polypeptide or fragment is expressed in a host transformedwith a recombinant DNA molecule according to the subject invention. Forexample, expression of a protein or peptide may be controlled by anypromoter/enhancer element known in the art.

In the context of the instant invention, the terms polypeptide, peptideand protein are used interchangeably. Likewise, the terms analogue andhomologous are also used interchangeably. It should be understood thatthe invention does not relate to the polypeptides in natural form ornative environment. Peptides and polypeptides according to the inventionhave been isolated or obtained by purification from natural sources (ortheir native environment), chemically synthesized, or obtained from hostcells prepared by genetic manipulation (e.g., the polypeptides, orfragments thereof, are recombinantly produced by host cells).Polypeptides according to the instant invention may also containnon-natural amino acids, as will be described below.

“Analogues” or “homologous” polypeptides will be understood to designatethe polypeptides containing, in relation to the native polypeptide,modifications such as deletion, addition, or substitution of at leastone amino acid, truncation, extension, or the addition of chimericheterologous polypeptides. Optionally, “analogues” or “homologous”polypeptides can contain a mutation or post-translational modifications.Among the “analogues” or “homologous” polypeptides, those whose aminoacid sequence exhibits 20.00% to 99.99% (inclusive) identity to thenative polypeptide sequence are preferred. The aforementioned range ofpercent identity is to be taken as including, and providing writtendescription and support for, any fractional percentage, in intervals of0.01%, between 50.00% and, up to, including 99.99%. These percentagesare purely statistical and differences between two polypeptide sequencescan be distributed randomly and over the entire sequence length.

“Analogues” or “homologous” polypeptide sequences exhibiting apercentage identity with the human 2-5 AS polypeptides, or subunitsthereof, can alternatively have 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99percent identity with the polypeptide sequences of the instantinvention. The expression equivalent amino acid is intended here todesignate any amino acid capable of being substituted for one of theamino acids in the basic structure without, however, essentiallymodifying the biological activities of the corresponding peptides and asprovided below.

By way of example, amino acid substitutions can be carried out withoutresulting in a substantial modification of the biological activity ofthe corresponding modified polypeptides; for example, the replacement ofleucine with valine or isoleucine; aspartic acid with glutamic acid;glutamine with asparagine; arginine with lysine; and the reversesubstitutions can be performed without substantial modification of thebiological activity of the polypeptides.

The subject invention also provides catalytically active fragments ofthe 2-5 AS polypeptide, and catalytically active fragments of the 2-5 ASsubunits, according to the invention, which are capable of eliciting animmune response against RSV. The immune response can provide components(either antibodies or components of the cellular immune response (e.g.,B-cells, helper, cytotoxic, and/or suppressor T-cells) reactive with thecatalytically active fragment of the polypeptide, the intact, fulllength, unmodified polypeptide, or both the catalytically activefragment of the polypeptide and the intact, full length, unmodifiedpolypeptides.

Catalytically active fragments according to the invention can comprisefrom five (5) amino acids to one amino acid less than the full length ofany polypeptide sequence provided herein. For example, fragmentscomprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, and up to one amino acid less than the full length aminoacid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, and SEQ ID NO:12, are provided herein.

Fragments, as described herein, can be obtained by cleaving thepolypeptides of the invention with a proteolytic enzyme (such astrypsin, chymotrypsin, or collagenase) or with a chemical reagent, suchas cyanogen bromide (CNBr). Alternatively, polypeptide fragments can begenerated in a highly acidic environment, for example at pH 2.5. Suchpolypeptide fragments may be equally well prepared by chemical synthesisor using hosts transformed with an expression vector containing nucleicacids encoding polypeptide fragments according to the invention. Thetransformed host cells contain a nucleic acid and are cultured accordingto well-known methods; thus, the invention allows for the expression ofthese fragments, under the control of appropriate elements forregulation and/or expression of the polypeptide fragments.

Modified polypeptides according to the invention are understood todesignate a polypeptide obtained by variation in the splicing oftranscriptional products of the 2-5 AS gene, genetic recombination, orby chemical synthesis as described below. Modified polypeptides containat least one modification in relation to the normal polypeptidesequence. These modifications can include the addition, substitution, ordeletion of amino acids contained within the polypeptides of theinvention.

Conservative substitutions whereby an amino acid of one class isreplaced with another amino acid of the same type fall within the scopeof the subject invention so long as the substitution does not materiallyalter the biological activity of the polypeptide. For example, the classof nonpolar amino acids include Ala, Val, Leu, Ile, Pro, Met, Phe, andTrp; the class of uncharged polar amino acids includes Gly, Ser, Thr,Cys, Tyr, Asn, and Gln; the class of acidic amino acids includes Asp andGlu; and the class of basic amino acids includes Lys, Arg, and His. Insome instances, non-conservative substitutions can be made where thesesubstitutions do not significantly detract from the biological activityof the polypeptide.

In order to extend the life of the polypeptides of the invention, it maybe advantageous to use non-natural amino acids, for example in the Dform, or alternatively amino acid analogs, such as sulfur-containingforms of amino acids. Alternative means for increasing the life ofpolypeptides can also be used in the practice of the instant invention.For example, polypeptides of the invention, and fragments thereof, canbe recombinantly modified to include elements that increase the plasma,or serum half-life of the polypeptides of the invention. These elementsinclude, and are not limited to, antibody constant regions (see forexample, U.S. Pat. No. 5,565,335, hereby incorporated by reference inits entirety, including all references cited therein), or other elementssuch as those disclosed in U.S. Pat. No. 6,319,691; 6,277,375; or5,643,570, each of which is incorporated by reference in its entirety,including all references cited within each respective patent.Alternatively, the 2-5 AS polynucleotides, or catalytically activefragments thereof, used in the instant invention can be recombinantlyfused to elements that are useful in the preparation of immunogenicconstructs for the purposes of vaccine formulation or elements usefulfor the isolation of the polypeptides of the invention.

The polypeptides, fragments, and immunogenic fragments of the inventionmay further contain linkers that facilitate the attachment of thefragments to a carrier molecule for delivery or diagnostic purposes. Thelinkers can also be used to attach fragments according to the inventionto solid support matrices for use in affinity purification protocols. Inthis aspect of the invention, the linkers specifically exclude, and arenot to be considered anticipated, where the fragment is a subsequence ofanother peptide, polypeptide, or protein as identified in a search ofprotein sequence databases as indicated in the preceding paragraph. Inother words, the non-identical portions of the other peptide,polypeptide, or protein is not considered to be a “linker” in thisaspect of the invention. Non-limiting examples of “linkers” suitable forthe practice of the invention include chemical linkers (such as thosesold by Pierce, Rockford, Ill.), peptides that allow for the connectionof the immunogenic fragment to a carrier molecule (see, for example,linkers disclosed in U.S. Pat. Nos. 6,121,424; 5,843,464; 5,750,352; and5,990,275, hereby incorporated by reference in their entirety). Invarious embodiments, the linkers can be up to 50 amino acids in length,up to 40 amino acids in length, up to 30 amino acids in length, up to 20amino acids in length, up to 10 amino acids in length, or up to 5 aminoacids in length.

In other specific embodiments, the 2-5 AS polypeptide or 2-5 AS subunitpolypeptide, peptides, derivatives, or analogs thereof may be expressedas a fusion, or chimeric protein product (comprising the protein,fragment, analog, or derivative joined via a peptide bond to aheterologous protein sequence (e.g., a different protein)). Such achimeric product can be made by ligating the appropriate nucleic acidsequences encoding the desired amino acid sequences to each other bymethods known in the art, in the proper coding frame, and expressing thechimeric product by methods commonly known in the art (see, for example,U.S. Pat. No. 6,342,362, hereby incorporated by reference in itsentirety; Altendorf et al. [1999-WWW, 2000] “Structure and Function ofthe F_(o) Complex of the ATP Synthase from Escherichia Coli,” J. ofExperimental Biology 203:19-28, The Co. of Biologists, Ltd., G. B.;Baneyx [1999] “Recombinant Protein Expression in Escherichia coli,”Biotechnology 10:411-21, Elsevier Science Ltd.; Eihauer et al. [2001]“The FLAG Peptide, a Versatile Fusion Tag for the Purification ofRecombinant Proteins,” J. Biochem Biophys Methods 49:455-65; Jones etal. [1995] J. Chromatography 707:3-22; Jones et al. [1995]“CurrentTrends in Molecular Recognition and Bioseparation,” J. Chromatography A.707:3-22, Elsevier Science B. V.; Margolin [2000] “Green FluorescentProtein as a Reporter for Macromolecular Localization in BacterialCells,” Methods 20:62-72, Academic Press; Puig et al. [2001] “The TandemAffinity Purification (TAP) Method: A General Procedure of ProteinComplex Purification,” Methods 24:218-29, Academic Press; Sassenfeld[1990]“Engineering Proteins for Purification,” TibTech 8:88-93; Sheibani[1999] “Prokaryotic Gene Fusion Expression Systems and Their Use inStructural and Functional Studies of Proteins,” Prep. Biochem. &Biotechnol. 29 (1):77-90, Marcel Dekker, Inc.; Skerra et al.[1999]“Applications of a Peptide Ligand for Streptavidin: TheStrep-tag”, Biomolecular Engineering 16:79-86, Elsevier Science, B. V.;Smith [1998] “Cookbook for Eukaryotic Protein Expression Yeast, Insect,and Plant Expression Systems,” The Scientist 12(22):20; Smyth et al.[2000] “Eukaryotic Expression and Purification of RecombinantExtracellular Matrix Proteins Carrying the Strep II Tag”, Methods inMolecular Biology, 139:49-57; Unger [1997] “Show Me the Money:Prokaryotic Expression Vectors and Purification Systems,” The Scientist11 (17):20, each of which is hereby incorporated by reference in theirentireties). Alternatively, such a chimeric product may be made byprotein synthetic techniques, e.g., by use of a peptide synthesizer.Fusion peptides can comprise polypeptides of the subject invention andone or more protein transduction domains, as described above. Suchfusion peptides are particularly useful for delivering the cargopolypeptide through the cell membrane.

Increasing the amount of 2-5 AS enzymatic activity (e.g., p40, p69,and/or p100 subunit activity) within a tissue is useful in preventing anRNA virus infection, or in treating an existing RNA virus infection.Thus, according to the methods of the subject invention, the amount of2-5 AS activity can be increased within a tissue by directlyadministering the 2-5 AS polypeptide or a catalytically active fragmentthereof to a patient suffering from or susceptible to an RNA virusinfection (such as exogenous delivery of the 2-5 AS p40, p69, and/orp100 subunit polypeptide) or by indirect or genetic means (such asdelivery of a nucleotide sequence encoding the 2-5 AS polypeptide or acatalytically active fragment thereof, or upregulating the endogenous2-5 AS polypeptide activity).

As used herein, the term “administration” or “administering” refers tothe process of delivering an agent to a patient, wherein the agentdirectly or indirectly increases 2-5 AS enzymatic function within thepatient and, preferably, at the target site. The process ofadministration can be varied, depending on the agent, or agents, and thedesired effect. Thus, wherein the agent is genetic material, such asDNA, the process of administration involves administering a DNA encoding2-5 AS, or a catalytically active fragment thereof, to a patient in needof such treatment. Administration can be accomplished by any meansappropriate for the therapeutic agent, for example, by parenteral,mucosal, pulmonary, topical, catheter-based, or oral means of delivery.Parenteral delivery can include for example, subcutaneous intravenous,intramuscular, intra-arterial, and injection into the tissue of anorgan, particularly tumor tissue. Mucosal delivery can include, forexample, intranasal delivery. According to the method of the presentinvention, a nucleotide sequence encoding the 2-5 AS or catalyticallyactive fragment is preferably administered into the airways of apatient, i.e., nose, sinus, throat, lung, for example, as nose drops, bynebulization, vaporization, or other methods known in the art. Oral orintranasal delivery can include the administration of a propellant.Pulmonary delivery can include inhalation of the agent. Catheter-baseddelivery can include delivery by iontropheretic catheter-based delivery.Oral delivery can include delivery of a coated pill, or administrationof a liquid by mouth. Administration can generally also include deliverywith a pharmaceutically acceptable carrier, such as, for example, abuffer, a polypeptide, a peptide, a polysaccharide conjugate, aliposome, and/or a lipid. Gene therapy protocol is also considered anadministration in which the therapeutic agent is a polynucleotidecapable of accomplishing a therapeutic goal when expressed as atranscript or a polypeptide into the patient. Further informationconcerning applicable gene therapy protocols is provided in the examplesdisclosed herein.

The pharmaceutical compositions of the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in a number of sourceswhich are well known and readily available to those skilled in the art.For example, Remington's Pharmaceutical Science (Martin E W [1995]Easton Pennsylavania, Mack Publishing Company, 19^(th) ed.) describesformulations which can be used in connection with the subject invention.Formulations suitable for parenteral administration include, forexample, aqueous sterile injection solutions, which may containantioxidants, buffers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and nonaqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze dried (lyophilized) conditionrequiring only the condition of the sterile liquid carrier, for example,water for injections, prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powder, granules, tablets,etc. It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations of the subject inventioncan include other agents conventional in the art having regard to thetype of formulation in question.

Therapeutically effective and optimal dosage ranges for 2-5 AS orcatalytically active fragments thereof can be determined using methodsknown in the art. Guidance as to appropriate dosages to achieve ananti-viral effect is provided from the exemplified assays disclosedherein.

As used herein, the term “catalytic activity” with respect to fragments,analogues, and homologs of the 2-5 AS polypeptide, or to fragments,analogues, and homologues of nucleotide sequences encoding the 2-5 ASpolypeptide, refers to 2′-5′ oligoadenylate synthetase activity. As usedherein, “2′-5′ oligoadenylate synthetase activity” refers topolymerization of ATP to produce 2′-5′ linked oligoadenylates, which inturn, activate a latent ribonuclease, RNase L, that degrades RNAs (see,for example, Katze et al., Nat. Rev. Immunol., September 2002, 2(9):675-687; Justesen et al., Cell Mol. Life. Sci., 57:1593-1612, 2000;Hartmann et al., J. Bio. Chem., 273(6):3236-3246, 1998; U.S. Pat. No.5,766,864). Preferably, the catalytic activity is an amount effective toinhibit RNA virus infection (pre-infection or post-infection). 2′-5′oligoadenylate synthetase activity can be determined directly orindirectly in vivo, or in vitro, using methods known in the art. Thus,cell-based assays can be utilized to determine whether an agent, such asa nucleotide sequence or polypeptide, exhibits the relevant catalyticactivity, and can be utilized to carry out the method of RNA virusinhibition of the subject invention.

RNA virus infections that can be inhibited using the present inventioninclude those that must produce double-stranded RNA as an intermediatestep in viral replication and those viruses for which interferon canactively inhibit viral replication. These RNA viruses can includedsingle-stranded or double-stranded RNA viruses, and have genomes ofpositive (+) or negative (−) strand polarity.

The present invention further provides methods of making the host cells,pharmaceutical compositions, and vectors described herein by combiningthe various components using methods known in the art.

The term “patient”, as used herein, refers to any vertebrate species.Preferably, the patient is of a mammalian species. Mammalian specieswhich benefit from the disclosed methods of treatment include, and arenot limited to, apes, chimpanzees, orangutans, humans, monkeys;domesticated animals (e.g., pets) such as dogs, cats, guinea pigs,hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sealions, elephant seals, otters, porpoises, dolphins, and whales. Human ornon-human animal patients can range in age from neonates to elderly. Thenucleotide sequences and polypeptides can be administered to patients ofthe same species or from different species. For example, mammalian,homologs can be administered to human patients.

The terms “2-5 AS p40 subunit” and “2-5 AS p40 subunit polypeptide” areused herein interchangeably to refer to the 2′-5′ oligoadenylatesynthetase p40 subunit gene or its coding sequence (CDS), itspolypeptide product, or a catalytically active fragment or analogue ofthe polypeptide product, and includes 2-5 AS p40 subunit peptidehomologs (such as mammalian orthologs (e.g., SEQ ID NOs: 11 and 12);NCBI Accession Number M33863) and isoforms, unless otherwise noted.Thus, the term includes all splice variants of the p40 subunit, such asthe 40 kDa (SEQ ID NOs: 1 and 2), 42 kDa, and 46 kDa (SEQ ID NOs:3 and4) splice variants of the 2-5 AS p40 subunit (NCBI Accession NumberNM_(—)016816).

The terms “2-5 AS p69 subunit” and “2-5 AS p69 subunit polypeptide” areused herein interchangeably to refer to the 2′-5′ oligoadenylatesynthetase p69 subunit gene or its coding sequence (CDS), itspolypeptide product, or a catalytically active fragment or analogue ofthe polypeptide product, and includes 2-5 AS p69 subunit peptidehomologs (such as mammalian orthologs) and isoforms, unless otherwisenoted. Thus, the term includes all splice variants of the p69 subunit,such as the 69 kDa (SEQ ID NOs:5 and 6) and 71 kDa (SEQ ID NOs:7 and 8)splice variants of the 2-5 AS p69 subunit (NCBI Accession NumberNM_(—)002535).

The terms “2-5 AS p100 subunit” and “2-5 AS p100 subunit polypeptide”are used herein interchangeably to refer to the 2′-5′ oligoadenylatesynthetase p100 subunit gene or its coding sequence (CDS) (SEQ ID NO:9),its polypeptide product (SEQ ID NO:10), or a catalytically activefragment or analogue of the polypeptide product, and includes 2-5 ASp100 subunit peptide homologs (such as mammalian orthologs) andisoforms, unless otherwise noted. Thus, the term includes all splicevariants of the p100 subunit (NCBI Accession Number AF063613).

The terms “comprising”, “consisting of”, and “consisting essentially of”are defined according to their standard meaning and may be substitutedfor one another throughout the instant application in order to attachthe specific meaning associated with each term.

Materials and Methods

Epithelial Cell Culture, Virus Infection and Plaque Assay. The HEp-2(ATCC CCL-23) cell line was obtained from the American Type CultureCollection (Manassas, Va.) and was maintained in Minimum Essentialmedium with Hank's salts (MEM) supplemented with 5% fetal bovine serum(FBS) (ATLANTA BIOLOGICALS, Norcross, Ga.) at 37° C. with 5% CO₂.Respiratory syncytial virus (RSV) A2 strain was obtained from ATCC(VR-1302) and was propagated in HEp-2 cells grown in MEM with 2% FBS ona monolayer culture. Viral stocks were prepared from infected HEp-2cells 5 days post infection (p.i.), stored at −70° C. in aliquots andused as the viral inoculum. RSV titers were quantified by plaque assayas described earlier (21).

MTT Cytotoxicity Assay. The effect of IFN-γ on the viability of cellswas determined using a MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (SIGMA,St. Louis, Mo.) cytotoxicity assay. Triplicate sets of cell monolayerswere used for each IFN-γ dose tested and for each time point. In thissystem, the mitochondrial dehydrogenase enzymes of living cells cleavethe tetrazolium ring of the yellow MTT to form purple formazan crystals,which are insoluble in aqueous solutions. The crystals were dissolved inacidified isopropanol, and the absorbance of the resulting purplesolution was spectrophotometrically measured at 540 nm. An increase ordecrease in the viable cell number results in a concomitant change inthe amount of formazan formed, indicating the degree of cytotoxicitycaused by the indicated dose of IFN-γ.

Immunoblot Analysis. IFN-γ treated cells were washed in cold PBS, pH 7.4and scraped into PBS at various time points. The cells were collected bycentrifugation at 6000 rpm for 3 min at 4° C. and the cell pellet wassuspended in a 2-pack volume of cell lysis buffer (50 mM Tris-HCl,pH7.4; 1% NP-40; 150 mM NaCl; 1 mM EGTA; 1 mM PMSF; 1 mg/ml aprotinin,leupeptin, pepstatin) and vortexed thoroughly. The cell lysate was spunat 13,000 rpm for 15′ at 4° C. to remove cellular debris. Thesupernatant was collected and the protein content estimated using theBCA (bicinchoninic acid) assay (PIERCE, Rockford, Ill.). 30 mg of totalprotein was mixed with an equal volume of 2×SDS sample buffer (22) andloaded onto a 10% SDS-PAGE and run at a 30 mA constant current for 2 to2.5 hours. For the detection of iNOS, the lysate of the IFN-γ andLPS-stimulated murine macrophage (RAW 264.7) was loaded onto the gel asa positive control. The proteins were transferred to a nitrocellulosemembrane overnight at a 12 mA constant current in transfer buffer (39 mMglycine, 48 mM Tris-HCl, 20% methanol) at 4° C.

Following protein transfer to the nitrocellulose membrane, the blotswere immediately placed into blocking buffer (5% non-fat dry milk, 10 mMTris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) and incubated for 30′ atroom temperature. The blots were then individually incubated overnightwith mAbs to IRF-1, IRF-2, PKR, cytokeratin-18 (SANTA CRUZ BIOTECHNOLOGYInc, Santa Cruz, Calif.), iNOS (TRANSDUCTION LABORATORIES, Lexington,Ky.) and phospho-eIF-2a (CELL SIGNALING, Beverly, Mass.) at 4° C. Blotswere washed three times in washing buffer (10 mM Tris-HCl, pH 7.5, 100mM NaCl, 0.1% Tween 20) and were subsequently incubated with anti-mouseIgG HRP conjugate (BOEHRINGER MANNHEIM, Indianapolis, Ind.) (1:5000) for30′ at room temperature. The blots were again washed in washing bufferand developed by the addition of ECL chemiluminescent detection reagents(0.125 ml/cm2) according to the manufacturer's instructions (AMERSHAMLIFE SCIENCES, Arlington Heights, Ill.). The blots were wrapped in saranwrap and exposed to Kodak X-OMAT AR films (EASTMAN KODAK, Rochester,N.Y.).

Nitrite Assay. Nitrite, a stable breakdown product of NO inphysiological systems, was assayed using the Griess reaction (23). Cellculture supernatants (100 μL) were added in triplicates to 100 μL ofGriess reagent (sulfanilamide 1%, naphthylethylenediaminedihydrochloride 0.1%, phosphoric acid 2.5%) using 96-well plates (SIGMA,St. Louis, Mo.). After incubation at room temperature for 10 min,absorbance at 550 nm was measured. A doubling dilution of a 50 M sodiumnitrite solution was used to generate a standard curve. The lower limitof the standard curve was 0.25 μM.

Northern Analysis. Northern blot analysis was performed to examine themRNA expression profile of IFN-γ-induced genes. Total cellular RNA wasisolated from cells using TRIZOL reagent (Life Technologies,Gaithersburg, Md.) following the manufacturer's instructions. Probes fornorthern hybridization were prepared by RT-PCR using gene specificprimers for IRF-1 (nucleotides 7-359), 2-5 AS p40 (nucleotides 2-492),2-5 AS p69 (nucleotides 21-503), RSV G (nucleotides 4688-5584), RSV F(nucleotides 5661-7385) and glyceraldehyde 3 phosphate dehydrogenase(GAPDH) (nucleotides 1-360). The PCR products were confirmed bysequencing. The probes were labeled using BrightStar Psoralen-Biotinlabeling kit (AMBION, Austin, Tex.) following manufacturer's protocol.10 mg of total RNA was size fractionated on 1% formaldehyde agarose gel,and transferred to nylon membranes (HYBOND N+, AMERSHAM, Piscataway,N.J.) using standard protocol (24) and cross-linked by UV irradiation(UV STRATALINKER 1800, STRATAGENE, San Diego, Calif.). Hybridization wascarried out at 42° C. overnight with 2-4 μM labeled probe and UltraHybhybridization solution (AMBION, Austin, Tex.).

The blots were washed twice with 2×SSC, 0.1% SDS for 5 minutes each andtwo more washes with 0.1×SSC, 0.1% SDS for 15 minutes each at 42° C. Theblots were processed for detection using the BRIGHTSTAR BIODETECT Kit(AMBION, Austin, Tex.) following manufacturer's protocol. The blots wereexposed to KODAK X-OMAT AR films (EASTMAN KODAK, Rochester, N.Y.) for1-15 minutes. The bands were quantified by using Advanced Quantifiersoftware (BIOIMAGE, Ann Arbor, Mich.) and the signals were normalizedwith the respective GAPDH signal.

Antisense Blocking of 2′-5′ Oligoadenylate Synthetase. Phosphorothioateantisense oligonucleotides (ODNs) were designed against p40 and p69subunits of 2′-5′ oligoadenylate synthetase. The sequences of antisenseODNs are as follows: p40 subunit, 5′-TTT CTG AGA TCC ATC ATT GA-3′ (SEQID NO: 17) and p69 subunit, 5′-TCC CCA TTT CCC ATT GC-3′ (SEQ ID NO:18). The control ODN sequences 5′-GTC TAT GAA TAC TTT CCT AG-3′ (SEQ IDNO: 19) and 5′-CAC CTC TAT CTC TCT CG-3′ (SEQ ID NO: 20) are a scrambleof the antisense sequence to p40 and p69 isomers, respectively, i.e.,identical in base composition. HEp-2 cells were treated with 1000 U/mlof IFN-γ protein for 20 hours. At the same time equimolar mixture ofantisense ODNs to both the isoforms of 2-5 AS or their scrambledmismatch ODNs were added at concentrations 0, 3, 30 and 90 nM. Cellswere infected with RSV at 20 h post-IFN-γ-treatment, as describedearlier. After 1 h of virus adsorption, cultures were supplemented withcomplete medium, which contained 1000 U/ml of IFN-γ and respectiveconcentrations of ODNs, and incubated for 72 hrs. ODNs were supplementedevery 8 hours. At 72 h pi, cells were washed three times with cold PBS,pH 7.4, harvested and the clear cell homogenate was used for plaqueassay.

2-5 AS Assay. A 2-5 Assay was done following the method describedpreviously (Ghosh et al., J. Biol. Chem. 272:15452-15458, 1997).Briefly, 20 μl of reaction mixture containing the cell homogenate, 20 mMTris-HCl, pH 7.5, 20 mM magnesium acetate, 2.5 mM dithiothreitol, 5 mMATP, 5 μCi of [a-32P] ATP, and 50 μg/ml poly(I).poly(C) was incubatedfor 3 h at 30° C. The reaction was stopped by boiling for 3 min andcentrifuged, and was incubated for 3 h at 37° C. with 3 μl of 1 unit/μlcalf intestine alkaline phosphatase to convert the unreacted [a-32P]ATPto inorganic phosphate. Two μl of the sample were then spotted on apolyethyleneimine-cellulose thin layer chromatography plate and resolvedin 750 mM KH₂PO₄, pH 3.5. The 2-5 A formed was then quantified by usingAdvanced Quantifier software (BIOIMAGE, Ann Arbor, Mich.) and expressedas arbitrary units.

Generation of Stable Cell Line Overexpressing Rnase L Inhibitor. HumanRLI cDNA was amplified as KpnI-BamHI cassette and cloned in pcDNA3.1(INVITROGEN, Carlsbad, Calif.) by the standard procedure (Sambrook etal., Molecular Cloning: A Laboratory Manual, and ed., Cold Spring HarborLaboratory, NY, 1989). HEp-2 cells were transfected with 5 mg ofpcDNA3.1-RLI using lipofectine (LIFE TECHNOLOGIES, Gaithersburg, Md.).The empty pcDNA3.1 vector was used as a control. Stable transfectantswere selected by culturing the cells in the presence of G418 (LIFETECHNOLOGIES, Gaithersburg, Md.). Individual clones were isolated andanalyzed for the expression of RLI mRNA. The clone that expressed RLI atthe highest level and had a normal morphology and growth pattern wasselected and named RLI-14.

RNAse L Assay. An RNAse L assay was done by ribosomal RNA cleavage assay(Player et al., Methods, 15:243-253, 1998). Briefly, cells wereharvested in NP-40 lysis buffer (10 mM HEPES, pH 7.5, 90 mM KCl, 1 mMmagnesium acetate, 0.5% (v/v) NP-40, 2 mM 2-mercaptoethanol, 100 mg/mlleupeptin) and S10 lysate was prepared and protein content was estimatedusing the BCA (bicinchoninic acid) assay (Pierce, Rockford, Ill.).Ribosomal RNA cleavage by RNAse L was assayed in a 20 ml reactioncontaining 200 mg protein, 2 ml of 10× cleavage buffer (100 mM HEPES, pH7.5, 1 mM KCl, 50 mM magnesium acetate, 10 mM ATP, 0.14 M2-mercaptoethanol), 100 nM 2′-5′A and incubated at 30° C. for 2 h. RNAwas isolated from the reaction using TRIZOL reagent (LIFE TECHNOLOGIES,Gaithersburg, Md.) following the manufacturer's instructions. 2 mg ofRNA was separated on agarose gel electrophoresis and the rRNA cleavageproducts were compared.

Animals. Female 6-8 weeks old wild type and STAT4^(−/−) BALB/c mice fromJackson Laboratory (Bar Harbor, Me.) were maintained in pathogen freeconditions at the animal center at USF College of Medicine. Allprocedures were reviewed and approved by the committee on animalresearch at the University of South Florida College of Medicine.

Cloning and recombination of adenoviral vectors. Murine 25 AS (p40) cDNAwas cloned into adenoviral transfer vector pShuttle-CMV (STRATAGENE, CA)at KpnI and XhoI sites. The left and right arms of pShuttle-CMV vectorcontains Ad5 nucleotides 34,931-35,935 and 3,534-5,790, which mediatehomologous recombination with pAdEasy-1 vector in E. coli, plus invertedterminal repeat (ITR) and packaging signal sequences (nucleotides 1-480of Ad5) required for viral production in mammalian cells. pAdEasy-1adenoviral plasmid (STRATAGENE, CA) contains all Ad5 sequences exceptnucleotides 1-3,533 (encompassing the E1 gene) and nucleotides28,130-30,820 (encompassing E3).

For generation of recombinant adenovirus plasmid, pShuttle-CMV-p40/LacZplasmids were linearized with PmeI and co-transformed with pAdEasy-1plasmid into recombination proficient BJ5183 cells. The recombinationwas confirmed by PacI digestion. The recombined clones werere-transformed into DH5α cells for large-scale plasmid purification.

Generation and purification of recombinant adenovirus. HEK293 cells,which produce the deleted E1 genes in trans, were transfected with 4 μgof PacI digested recombinant adenovirus plasmid DNA with lipofectin(LIFE TECHNOLOGIES, MD). Cells were harvested 7-10 dayspost-transfection, resuspended in PBS and recombinant virus wascollected by 3-4 freeze-thaw cycles. The recombinant virus expressingmurine p40 and LacZ were termed Ad-p40 and Ad-LacZ, respectively. Theviruses were amplified by infecting fresh HEK-293 cells. Viruses werefurther purified by CsCl banding (Becker et al., Methods Cell Biol., 43Pt. A:161-189, 1994). The viral band was extracted and CsCl was removedby passing through Centricon-100 columns (MILLIPORE, MA).

Quantitation of RSV titers in lung. To quantify RSV titers in the mouselung whole lungs were first weighed and placed immediately in EMEM mediasupplemented with 10% FBS. Lungs were homogenized, centrifuged at 10,000RPM for 10 minutes at 4° C., the clear supernatants were used for plaqueassays by shell vial technique (Kumar et al., 2002).

Pulmonary Function. To evaluate the pulmonary function in vaccinated andcontrol groups, mice were administered IGT, as described earlier. Threedays later, airway responsiveness was assessed non-invasively inconscious, unrestrained mice with a whole body plethysmograph (BUXCOELECTRONICS, Troy, N.Y.), as previously described (Matsuse et al., J.Immunol. 164:6583-6592, 2000). With this system, the volume changes thatoccur during a normal respiratory cycle are recorded as the pressuredifference between an animal-containing chamber and a reference chamber.The resulting signal is used to calculate respiratory frequency, minutevolume, tidal volume, and enhanced pause (Penh). Penh was used as themeasure of bronchoconstriction and was calculated from the formula:Penh=pause×(peak expiratory pressure/peak inspiratory pressure), wherepause is the ratio of time required to exhale the last 30% of tidalvolume relative to the total time of expiration. Mice were placed in theplethysmograph and the chamber was equilibrated for 10 min. They wereexposed to aerosolized PBS (to establish baseline) followed byincremental doses (6, 12.5, 25, and 50 mg/ml) of methacholine (SIGMACHEMICALS, St. Louis, Mo.). Each dose of methacholine was aerosolizedfor 5 min, and respiratory measurements were recorded for 5 minafterward. During the recording period, an average of each variable wasderived from every 30 breaths (or 30 s, whichever occurred first). Themaximum Penh value after each dose as used to measure the extent ofbronchoconstriction.

Bronchoalveolar lavage (BAL) and histology of the lung. Bronchoalveolarlavage were performed on Ad-p40 administered and control mice, asdescribed before (Kumar et al., 1999). Histological staining and asemiquantitative analysis of airway inflammation from the lungs of p40treated and control groups of mice were performed, as described earlier(Kumar et al., 1999). Lung inflammation was assessed after staining thesections with hematoxylin and eosin (HE). The entire lung section wasreviewed, and pathological changes were evaluated for epithelial damage,peribronchovascular cell infiltrate, and interstitial-alveolar cellinfiltrate for the mononuclear cells and polmorphonuclear cells.

Statistical Analysis. Experiments were repeated 2 to 4 times for eachexperiment as indicated. Statistical significance was analyzed usingpaired two-tailed student's t-test. Differences were consideredstatistically significant when the p-value was less than 0.05.

Example 1 IFN-γ Attenuates RSV Infection in Human Epithelial Cells

To examine the effect of IFN-γ on RSV infection, HEp-2 cells werepre-incubated for 4-20 h with different concentrations of IFN-γ andsubsequently infected with RSV. Respective concentrations of IFN-γ wereadded back to the cells in complete medium after the removal of viralinoculum. Cells were harvested at 72 h p.i., and viral titer wasdetermined by plaque assay. RSV replication was inhibited significantlywith the addition of various concentrations of IFN-γ to both cell linesprior to RSV infection (FIGS. 1A and B). A 97% inhibition of replicationwas observed in HEp-2 and A549 cells, at 1000 U/ml of IFN-γ added at 20h pre-infection. Cells treated with IFN-γ 4 h pre-infection also showedsignificant reduction (p<0.01) in RSV titer (50% reduction). Asignificant decrease (p<0.01) in RSV titer (39% reduction) was observedin A549 cells, which were not treated with IFN-γ before infection, butwere only treated at 1 h post infection (FIG. 1C). To rule out thepossibility that the reduction in RSV titers could be due tocytotoxicity of IFN-γ, a MTT cytotoxicity assay was performed. Theresults indicate that the cells were metabolically as viable as theuntreated control cells when treated with the highest concentrations ofIFN-γ (1,000 U/ml; FIG. 1D). Thus, IFN-γ did not exhibit any cytotoxicor growth inhibitory effect on these cells. These results suggest thatthe treatment of cells with soluble IFN-γ results in a significantdecrease in RSV infection in epithelial cells.

Example 2 IFN-γ Induces IRF-1 Protein Expression

ISGs implicated in the antiviral activity of IFNs include IRFs, doublestranded RNA activated protein kinase (PKR) and inducible nitric oxidesynthase (iNOS). To identify the ISGs in these cells potentiallyinvolved in protection against RSV infection, proteins were analyzedfrom cells at various time points post treatment with IFN-γ (1000 U/ml).A western blot analysis was performed using specific antibodies toIRF-1, IRF-2 and PKR (FIG. 2A). There was increased expression of IRF-1but no change in the expression of IRF-2 following IFN-γ addition.Expression of IRF-1 increased after 30′ of IFN-γ addition. Theexpression of PKR decreased gradually over time (FIG. 2B) and no changein the expression of phospho-eIF-2a was observed following IFN-γaddition. Cytokeratin-18 was used as an internal control, the expressionof which did not change with the addition of IFN-γ. To examine if IFN-γinduced iNOS plays a role in antiviral action, iNOS expression wasexamined by western blotting (FIG. 2B). The expression of iNOS proteincould not be detected before and after IFN-γ addition. Murine macrophagecell lysate containing iNOS was used as a positive control, which didnot bind to the cytokeratin-18 antibody used as internal control. Torule out completely the involvement of iNOS in the antiviral effect ofIFN-γ, the level of nitric oxide (NO) was examined in the culturesupernatant of both HEp-2 and A549 cells before and after the additionof IFN-γ at various time points. No detectable level of NO (lowestconcentration of standard was 0.25 mM) was observed in both cell linesat any time point, i.e., before or after IFN-γ addition in both celllines. A similar expression pattern was observed for IRF1, IRF2, PKR andiNOS in A549 cells. These results indicate that IFN-γ up-regulates IRF-1in these cells and neither PKR nor iNOS play any role in the antiviralactivity of IFN-γ against RSV infection in human epithelial cell lines.

Example 3 Exogenous IFN-γ Upregulates mRNA Synthesis of IRF-1 and 2-5 AS

IRF-1 has been reported to play a role in antiviral activity via theinduction and activation of 2-5 AS (Reis et al., EMBO J. 11:185-193,1992). Northern analysis was performed using gene specific probes forIRF-1 and the p40 and p69 isoforms of 2-5 AS (FIG. 3). The IRF-1 mRNAwas induced at 30 min after addition of IFN-γ and continued to increasegradually thereafter until 48 h post exposure. The induction of the p40and p69 isoforms of 2-5 AS was observed starting at 4 h and peaked at 24h post exposure. The p40 probe hybridized to two transcripts of 1.8 and1.6 kbp. Similarly, the p69 probe hybridized to four expectedtranscripts of 5.7, 4.5, 3.7 and 3.2 kbp of which 5.7 kbp was the majortranscript. These results suggest that IFN-γ induces IRF-1, which inturn, up regulates 2-5 AS, suggesting that the latter may be involved inthe anti-RSV mechanism of IFN-γ.

Example 4 2-5 as Antisense Oligonucleotides Abrogate the AntiviralEffect of IFN-γ in HEp-2 Cells

To investigate whether IFN-γ induced anti-RSV activity is mediated by2-5 AS, IFN-γ exposed (1000 U/ml at 20 h pre-infection) HEp-2 cells weretreated with equimolar mixture of antisense oligonucleotides (ODNs) toboth p40 and p69 isoforms of 2-5 AS. Scrambled mismatch of the antisenseODN sequence to p40 and p69 at the same concentration were used ascontrol. RSV infection was barely detectable in cells either treatedwith IFN-γ alone or with cells treated with IFN-γ and control ODNs butnot in those treated with IFN-γ and antisense ODNs, as shown in FIG. 4.Addition of antisense ODN significantly reverted (p<0.01) the antiviraleffect of IFN-γ against RSV infection and this reversal wasdose-dependent and increased with increasing concentrations of antisenseODNs. As shown in FIG. 4, 2-5 AS activity was reduced in a dosedependent manner in the cells treated with antisense ODN to 2-5 AS butnot control ODN. These results indicate that the addition of antisenseODNs to 2-5 AS to IFN-γ-treated cells reduced 2-5 AS activity in thesecells and in turn the antiviral effect of IFN-γ.

Example 5 Overexpression of RNAse L Inhibitor (RLI) does not Alter theIFN-γ Responses in HEp-2 Cells

In addition to RNAse L, RNAse L inhibitor (RLI) has been implicated inthe antiviral effect of IFN-γ. To determine the role of 2-5 A/RNaseL-mediated antiviral mechanism, a stable cell line expressing RLI,RLI-14, was established. A northern analysis of RNAs from RLI-14 andHEp-2 using gene specific probe for RLI showed a major 3.5 kb transcriptand a minor 2.8 Kb transcript (FIGS. 5A and 5B). A seven-fold increasein the major RLI transcript expression was observed in RLI-14 cells whencompared to HEp-2 cells. The analysis of IFN-γ induced proteins inRLI-14 cell line by western blotting showed that IFN-γ inducedexpression of IRF-1, but not IRF-2, at 30 min post induction and IRF-1expression continued to increase thereafter until 48 h (FIG. 5C) as inHEp-2 cells (FIG. 2A). Also, a time-specific decrease in PKR proteinconcentration was observed after IFN-γ addition in the RLI-14 cell line.The expression of cytokeratin-18, used as an internal control, remainedunchanged with IFN-γ addition. The level of mRNA expression of IRF-1,p40 and p69 isoforms of 2-5 AS was observed by northern analysis, andthe expression level showed a gradual increase over time following IFN-γstimulation (FIG. 5D) as in HEp-2 cells (FIG. 3). These results suggestthat overexpression of RLI does not change the expression pattern of theIFN-γ-induced genes involved in antiviral activity of these cells.

Example 6 RNase L Inhibitor (RLI) Overexpression Decreases the AntiviralActivity of IFN-γ

To examine the effect of the overexpression of the RNase L inhibitor,both HEp-2 and RLI-14 cells were treated with IFN-γ at 100-1000 U/ml at20 h pre-infection and subsequently infected with RSV. IFN-γ was addedback to the cells at respective concentrations following RSV infection.HEp-2 cells treated with 100 and 1000 U/ml of IFN-γ showed significantinhibition (p<0.001) of RSV infection (72% and 97% reduction,respectively) when compared to untreated cells. In marked contrast,RLI-14 cells showed significantly lower inhibition of infection (only12% and 22% reduction, respectively) compared to HEp-2 cells atrespective concentrations of IFN-γ, as showed in FIG. 6. In absence ofIFN-γ treatment, both cell lines exhibited identical RSV titers uponinfection. However, the viral titer significantly decreased (p<0.01)when the concentration of IFN-γ was increased from 100 U/ml to 1000 U/mlin RLI-14 cells. This demonstrates that increase in IFN-γ led to higherexpression of 2-5 AS and in turn production of 2-5 A, which subsequentlybound to RNase L and increased the level of active RNase L by releasingRNase L from its inactive complex. Reduction in virus replication wasinhibited in RLI-14 cells (%) when compared to HEp-2 cells (%), as shownin FIG. 6. In order to examine whether the reduction in inhibition ofRSV infection in RLI-14 cells was due to reduced RNAse L activity inthese cells, RNAse L assay was done using ribosomal RNA cleavage assay.This reaction uses cell lysate as a source of both substrate and enzyme,thus giving a comparison of the ribonuclease activity of RNAse L indifferent cell types. The results confirm that ribonuclease activity ofRNAse L is indeed reduced in RLI-14 cells when compared to HEp-2 cellsas evident from the rRNA cleavage products, as shown in FIG. 6.Together, these results confirm the involvement of 2-5 A/RNase L in theantiviral effect of IFN-γ against RSV infection.

The finding that treatment of HEp-2 and A549 cells at 20 h pre-infectionwith as low as 100 U/ml of IFN-γ proteins inhibits RSV infection andreplication when compared to untreated cells, has significanttherapeutic implications. HEp-2 and A549 cells treated with 1000 U/ml ofIFN-γ at 20 h pre-infection exhibited a 97% (30-31 fold in log 10PFU/ml) reduction in RSV titer. The RSV titer also decreased by 39% (1.7fold reduction in log 10 PFU/ml) in these cells, which were not treatedwith IFN-γ prior to infection but were only treated immediately afterRSV infection. RSV is resistant to the antiviral effects of type-Iinterferons and human MxA. It has been reported that overexpression ofIFN-γ by gene transfer and by recombinant RSV attenuates RSV replicationin a mouse model of RSV infection. However, the mechanism of antiviralaction of IFN-γ against RSV is not known.

The elucidation of the mechanism underlying IFN-γ-mediated resistance toRSV infection in human epithelial cells has been the main focus of thisinvention. The mechanism of antiviral action of IFN-γ is complex and maybe unique for individual cell lines and viruses. A profile of ISGs,relevant to antiviral activity in these epithelial cells, establish thatIFN-γ exposure results in induction of both the mRNA and protein forIRF-1 but not IRF2. In non-induced cells the IRF-2 protein functions asa repressor of ISGs. IFN-γ induction temporarily removes this repressionand activates ISGs including IRF-1. IRF-1 and IRF-2 compete for the samecis acting recognition sequences but with opposite effects. Findings inthese epithelial cells are consistent with those found for humanmacrophages, where IFN-γ treatment does not enhance IRF-2 geneexpression, despite strong upregulation of IRF-1 mRNA expression. Twoadditional ISGs, PKR and iNOS proteins were examined for their role inIFN-γ induced antiviral activity. IFN-γ activates PKR, which in turnphosphorylates and inactivates eukaryotic initiation factor-2a (eIF-2a)and leads to restriction of cellular as well as viral protein synthesis.The iNOS is also known to mediate antiviral property of IFN-γ. However,a time specific decrease in PKR expression and no change inphosphorylation of eIF-2a and the lack of detectable levels of iNOSprotein or NO in IFN-γ-stimulated HEp-2 and A549 cells indicate thatneither PKR and phospho-eIF-2a nor iNOS play any role in IFN-γ mediatedinhibition of RSV infection in these cells.

To further dissect the mechanism of IFN-γ mediated anti-RSV activity inHEp-2 and A549 cells, IRF-1 induced expression of 2-5 AS was examined.Of the four isoforms (p40, p46, p69, and p100) of 2-5 AS detected inhuman cells to date, the expression pattern of the p40 and p69 isoformsfollowing IFN-γ stimulation was examined in this study because of thefollowing. The p40 and p46 isoforms of 2-5 AS, which are dependent ondsRNA for activation, are derived from the same gene by differentialsplicing between the fifth and an additional sixth exon of this gene andare thus identical for the first 346 residues, except for theirC-terminal ends. Of the two high molecular weight isoforms, p69, but notp100, requires dsRNA for activation. The expression of 2-5 AS p40 andp69 are induced by IFN-γ in these cells at 4 h and peaks at 24 h postIFN-γ addition. Therefore, the antiviral effect of IFN-γ in these cellsis observed when the cells are treated with IFN-γ at 4 h pre-infectionand is highest when treated at 20 h pre-infection as the level of 2-5 ASis at peak at that time. These data suggest that the antiviral mechanismof IFN-γ against RSV infection is mediated by the activation of IRF-1,which in turn activates the 2-5 AS system. A dose-dependent abrogationof 2′-5′ AS activity and in turn the anti-RSV effect of IFN-γ by theaddition of an equimolar mixture of antisense ODNs to p40 and p69, butnot by the scrambled mismatch ODNs, provide evidence supporting the roleof 2-5 AS in the antiviral mechanism of IFN-γ against RSV infection.

2-5 AS induces 2-5 A, which binds to and activate RNase L, which cleavesdouble stranded RNA 3′ of UpN residues. The levels of RNase L areincreased in growth-arrested cells and following IFN-γ treatment;however, its biological activity is thought to be controlled at thelevel of enzymatic activation rather than through regulation of itstranscription and translation. Increasing endogenous levels of 2-5 Aleads to enhanced RNase L activity, which suggests that intracellularlevels of 2-5 A are rate limiting in the activation of RNase L, whereascellular levels of RNase L are sufficient for maximal biologicalactivity. Furthermore, RNase L remains in an inactive form in the cellsbeing bound to an inhibitor, RLI, which codes for a 68 kDa protein whosemRNA is not regulated by IFN-γ. RLI induces neither 2-5 A degradationnor reversible modification of RNase L when expressed in a reticulocytelysate, but antagonizes the binding of 2-5 A to RNase L, thus, itsnuclease activity, since 2-5 A binding is a prerequisite to RNase Ldimerization and activation.

RLI-14, a stable cell line overexpressing RLI, was established fromHEp-2 cells and characterized to determine precisely the involvement ofRNase L in the antiviral mechanism of RSV infected epithelial cells. Thefinding that RLI-14 was almost identical to the parent HEp-2 cells inits response to IFN-γ shows that RLI overexpression does not alter theinduction of ISGs in these cells (FIGS. 5A-D). Nonetheless, reducedRNAse L activity and antiviral activity of IFN-γ in RLI-14 cells (FIG.6), confirmed that the RNase L activity is indeed critical to theantiviral effect of IFN-γ and is only partly controlled by the elevatedlevels of 2-5 AS in these cells following IFN-γ treatment. The reductionin antiviral effect of IFN-γ in these cells was dependent on the dose ofIFN-γ, indicating that the level of 2-5 A, which is regulated by IFN-γand the level of RLI are crucial in determining which pathway cells willfollow. The importance of the level of 2-5 A was confirmed bypreliminary findings which showed significant reduction in RSV infectionwhen HEp-2 cells were treated with 100 U/ml of IFN-γ 20 h pre-infectionand transfected with 1 mM 2-5 A 2 h pre-infection, when compared to thecells treated with 100 U/ml of IFN-g alone. Similarly the importance ofthe level of RLI in the antiviral activity was reported for HIV, whereRLI is induced during HIV1 infection and down regulates the 2-5 A/RNaseL pathway in human T cells.

In summary, these results demonstrate that IFN-γ inhibits RSV infectionof human epithelial cells. Specifically, in HEp-2 and A549 epithelialcells, IFN-γ upregulates IRF-1, which in turn, induces 2-5 AS. Further,the 2-5 AS generates 2-5 A that activates RNase L, which is normallyfound in the cytoplasm in inactive state bound to RLI. Thus, RNaseL-mediated cleavage of viral RNA is governed by the ying-yang mechanisminvolving 2-5 A and RLI. In a 2-5 A-dominant state cells are protectedfrom RSV infection due to the activation of RNase L. In contrast, anRLI-dominant condition attenuates the antiviral effect by inactivationof RNase L. Since, 2-5 A and RLI are respectively, governed byIFN-γ-dependent and independent mechanisms, treatment with IFN-γ oroverexpression of 2-5 AS should provide an efficient means to redirectthe 2-5 A:RLI ratio toward a shift in favor of 2-5 A and achieve aprofound antiviral effect.

Example 7 2-5 AS Plasmid DNA Vaccination Attenuates RSV Infection andPathogenesis

As shown in FIG. 8A, 2-5 AS pDNA vaccine decreases lung RSV titers.BALB/c mice (n=4) were intranasally administered with p2′-5′ AS (25 mgof DNA each time complexed with lipofectamine) or an equal amount ofempty pVAX vector DNA 3 times in 2-day intervals. Mice were infectedwith RSV seven days after last DNA administration and were sacrificed onday 5 post-infection. BAL was performed and lungs were collected. RSVtiter was determined by plaque assay from the lung homogenate. Theresults show that 2-5 AS cDNA vaccination can significantly attenuatelung titers of RSV in infected mice.

FIG. 8B shows that reduction of viral titers is associated withreduction in MIP-1α. Expression level of MIP-1α was determined from lunghomogenate by ELISA. The results show that vaccination with 2-5 AS cDNAdecreases production of chemokine MIP-1α which is known to beproinflammatory in action.

In FIG. 8C, 2-5 AS overexpression increased the macrophage populationsignificantly compared to RSV infected mice. BAL cell differential wasperformed and percentages of macrophage, lymphocytes, and neutrophilswere determined. The results show that 2-5 AS does not alter thecellular composition of the lung. No significant changes are seen inlymphocytes and macrophages, however the percent of neutrophils isincreased in the lungs of mice treated with 2-5 AS cDNA. FIGS. 9A-9Cshow that 2-5 AS vaccination significantly decreased pulmonaryinflammation. Histological sections from lungs were stained withhematoxylin and eosin and representative photomicrographs are shown.

Example 8 AD-2-5 AS (P40) Decreases Lung RSV Titers

A reduction in virus titer is the gold standard by which theeffectiveness of an antiviral therapy is measured. Mice wereintranasally administered with 10⁸ PFU/ml rAD-p40 and then infected withRSV 4 h later. Analysis of lung virus titers following acute, live RSVinfection at day 5 post infection show a significant (100-fold, P<0.01)reduction in RSV titers in Ad-p40 treated mice compared to controls(FIG. 10). These results indicate that the rAD-p40 treatment constitutesan effective prophylaxis against RSV infection.

Example 9 AD-2-5 AS (P40) Decreases AHR in Mice

The safety of an antiviral therapy especially can be measured by adecrease in RSV-induced AHR. To test whether Ad-p40 administrationreduces RSV-induced airway hyperreactivity, the % baseline enhancedpause (Penh) was measured in a group of mice treated with rAD-p40 priorto RSV infection and their AHR was compared with untreated RSV infectedgroup. Mice receiving Ad-p40 exhibited a similar response tomethacholine challenge when compared to uninfected PBS control group(FIG. 10). These results suggest that the Ad-p40 induced decrease in RSVinfection decreases AHR.

Example 10 AD-2-5 AS (P40) Normalizes Cellular Infiltration to the Lung

The inflammation in the lung due to RSV infection is due to infiltrationinto the lung of a large number of macrophages and lymphocytes. Todetermine whether treatment with rAD-p40 affects migration of thesecells to the lung, mice administered with rAD-p40 and RSV infected werecompared to RSV infected mice without treatment and naïve mice ascontrol and to rAd-lacZ as control. Mice with p40 gene transfer and RSVinfection show a BAL cell differential similar to that of normaluninfected animals, lack of AHR compared to RSV-infected animals withoutp40 gene transfer and lack of the peribronchiolar and perivascularinflammation suggesting that intranasal p40 can potentially be aneffective anti-viral approach in vivo for RSV infection.

Example 11 AD-2-5 AS(P40) Decreases RSV Infection-Induced PulmonaryInflammation

Lung inflammation was examined in different groups of mice. Mice treatedwith Ad-p40 and Ad-lacZ upon acute RSV infection exhibit relatively lessdisruption of the epithelium and cellular infiltration. Representativepathological features reveal that groups of mice receiving Ad-p40exhibit less epithelial damage, and reduced mononuclear cell (MNC) andpolymorphonuclear cell (PMNC) infiltrates in the interstitial andperibronchovascular region, as compared to Ad-lacZcontrols (FIGS.12A-12H). These results suggest that the Ad-p40 protects mice from RSVinfection-induced pulmonary inflammation. These results suggest thatAd-p40 protects mice from RSV infection-induced pulmonary inflammation.

The finding that transient gene expression therapy can substantiallyreduce lung RSV titers by 2 logs (100-fold), inhibit RSV-infectioninduced AHR and make the lung resistant to inflammation by acute RSVinfection is very significant. These results suggest tremendoustherapeutic potential of this approach. The other members of this familyinclude the measles virus, the sendai virus, parainfluenza 1, 2, and 3,the mumps virus, the simian virus, and the newcastle virus. This findingis also important for other Paramyxoviruses, such as rotavirus thatcauses juvenile diarrhea in millions of children worldwide. Furthermore,beyond this family of viruses, the 2-5 AS/RNase L cascade has beenimplicated in the anti-viral activity of picorna viruses, (Hassel, B Aet al. Embo J, 1993, 12 (8):3297-304; Benavente, J et al. J. Virol.1984, 51 (3):866-71; Goswami, B B and Sharma, O K. J Biol Chem, 1984,259 (3):1371-4; Nilsen, T W et al. Mol Cell Biol, 1983, 3 (1):64-9),vaccinia virus (Maitra, R K and Silverman, R H. J Virol, 1998, 72(2):1146-52; Banerjee, R et al. Virology, 1990, 179 (1):410-5), reovirus(Kumar, R et al. J Virol, 1988, 62 (9):3175-81), HIV (Saito, H et al.Keio J Med, 1996, 45 (3):161-7; 45), EMCV (Glezen, W P et al. Am J DisChild, 1986, 140 (6):543-6), Hepatitis B and C virus (Groothuis, J R etal. Pediatrics, 1988, 82 (2):199-203; Nelson W E, Behrman R E, KliegmanR. Nelson Textbook of Pediatrics. 15 ed. Philadelphia).

Moreover, besides human disease, this finding may have implications, forRSV of cattle (BRSV), sheep, and goats. If 2-5 AS mediated approach issuccessful, the mortality and morbidity due to RSV infection can bereduced. Also, RSV has been linked with the development of asthma, andhence, prevention or successful treatment of RSV is expected to decreasethe incidence of asthma and fatal exacerbation of asthma due to RSV.Adults infected with RSV miss work for an average of 7-9 days as opposedthose with flu who miss an average of 6-7 days. Therapeutic treatmentcan reduce the number of absences from the work, which exceeds the fluinfection. Also, prophylaxis prior to and during viral season andtreatment immediately after infection can lead to a substantial decreasein hospitalization and emergency visits due to RSV infection.

RSV is one of the important viral respiratory pathogens that produce anannual epidemic of respiratory illness. In children, common diseasesassociated with RSV infection primarily include interstitial lungdiseases, such as bronchiolitis, and asthma. RSV is estimated to cause85% of the cases of acute bronchiolitis that affects infants and youngchildren (Shay, D K et al. Jama, 1999, 282 (15):1440-6). Some childrenmay become infected during three or four successive RSV seasons. Eachyear, RSV is responsible for up to an estimated 125,000 pediatrichospitalizations, with a mortality rate of approximately 2% (Heilman, CA. J Infect Dis 1990, 161 (3):402-6; Shay, D K et al. J Infect Dis,2001, 183 (1):16-22; Altman, C A et al. Pediatr Cardiol, 2000, 21(5):433-8; Simoes, E A. Lancet, 1999, 354 (9181):847-52; Falsey, A R. etal. J Infect Dis, 1995, 172 (2):389-94). Among hospitalized infants withchronic lung and heart disease, the RSV mortality rate may be as high as5%. Up to half of all pediatric admissions for bronchiolitis andone-quarter of admissions for pneumonia are due to RSV (La Via et al.,Clin. Pediatr. (Phila), 32 (8):450-454, 1993). RSV is a major riskfactor for a number of other health conditions, such asimmuno-deficiency, cardiac arrhythmia, congenital heart disease, andunusual atrial tachycardia (Donnerstein et al., J. Pediair. 125(1):23-28, 1994).

Emerging evidence also suggests that RSV is an important pathogen inprofusely healthy adults as well (Hall et al., Clin. Infect. Dis.33(6):792-796, 2001). In a study of 15 adults who were challenged by RSVafter a natural infection, 50% were reinfected after two months; by 26months 73% were reinfected (Fixler, D E. et al. Pediatr Cardiol, 1996,17 (3):163-8). RSV infection is also clinically important in previouslyhealthy working adults (Hogg, J C. et al. American Journal ofRespiratory & Critical Care Medicine, 1999, 160 (5):S26-S28). In thisstudy, of a total of 2960 18-60 year-old adults studied during 1975 to1995, 211 (7%) acquired RSV infection; 84% of infections weresymptomatic—74% upper respiratory tract infection, 26% lower respiratorytract infection and 40% were febrile. RSV is a major risk factor for thedevelopment and/or exacerbation of asthma and chronic obstructivepulmonary disorder (COPD), and about 30 million of Americans suffersfrom asthma and COPD.

The prevalence of bronchiolitis in infants as well as asthma and COPDhas increased throughout the world, including in the United States, overthe past two decades. The rates of death from asthma have increased from0.8 per 100,000 in 1977-78 to 2.0 per 100,000 in 1991, and these rateshave increased in almost all age groups in the United States (Sly, R M.Ann Allergy. 1994, 73 (3):259-68). Asthma is the most common cause ofthe admission of children to the hospital, and it is the most commonchronic illness causing absence from school and work in North America(Nelson, R P, Jr., et al. J Allergy Clin Immunol, 1996, 98 (2):258-63).The total costs of illnesses related to asthma in 1990 were 6.2 billion,a 53% increase in direct medical expenditures and a 23% increase inindirect costs since 1985 (Weiss, K B et al. N Engl J Med, 1992, 326(13):862-6). The total estimated cost in 1995 for the treatment ofallergic diseases, asthma, chronic sinusitis, otitis media, and nasalpolyps, was about $10 billion (Baraniuk, J N. J Resp Dis, 1996, 17(S11)). Together, these diseases lead to a significant reduction in thequality of life and a tremendous economic loss.

Finally, although studies with 2-5 AS (p40) in the adenovirus systemprovides the “proof of concept” for the anti-RSV activity, other virusvectors, including adeno-associated vectors (Zhao, N et al. MolBiotechnol, 2001, 19 (3):229-37; Monahan, P E et al. Mol Med Today,2000, 6 (11):433-40; Senior, K. Lancet, 2002, 359 (9313):1216) can beused to express this p-40 or other 2-5 AS gene(s) for the antiviralactivity.

Example 12 Gene Therapy

In the therapeutic and prophylactic methods of the present invention,the nucleotide sequence encoding 2-5 AS, or a catalytically activefragment thereof, can be administered to a patient in various ways. Itshould be noted that the vaccine can be administered as the compound oras pharmaceutically acceptable salt and can be administered alone or asan active ingredient in combination with pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles. In those cases in which theRNA virus is a virus that infects the patient's respiratory system, thecompounds can be administered intranasally, bronchially, via inhalationpathways, for example. The patient being treated is a warm-bloodedanimal and, in particular, mammals including man. The pharmaceuticallyacceptable carriers, diluents, adjuvants and vehicles as well as implantcarriers generally refer to inert, non-toxic solid or liquid fillers,diluents or encapsulating material not reacting with the activeingredients of the present invention.

It is noted that humans are treated generally longer than the miceexemplified herein, which treatment has a length proportional to thelength of the disease process and drug effectiveness. The doses may besingle doses or multiple doses over a period of several days, but singledoses are preferred.

The carrier for gene therapy can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity, when desired, can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Nonaqueous vehicles such a cottonseed oil, sesame oil, olive oil,soybean oil, corn oil, sunflower oil, or peanut oil and esters, such asisopropyl myristate, may also be used as solvent systems for compoundcompositions. Additionally, various additives that enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.According to the present invention, however, any vehicle, diluent, oradditive used would have to be compatible with the compounds.

Examples of delivery systems useful in the present invention include,but are not limited to: U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616;4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224;4,439,196; and 4,475,196. Many other delivery systems and modules arewell known to those skilled in the art.

A pharmacological formulation of the nucleotide sequence utilized in thepresent invention can be administered orally to the patient.Conventional methods such as administering the compounds in tablets,suspensions, solutions, emulsions, capsules, powders, syrups and thelike are usable. Known techniques which deliver the vaccine orally orintravenously and retain the biological activity are preferred.

In one embodiment, the nucleotide sequence can be administered initiallyby nasal infection to increase the local levels of 2-5 AS enzymaticactivity. The patient's 2-5 AS activity levels are then maintained by anoral dosage form, although other forms of administration, dependent uponthe patient's condition and as indicated above, can be used. Thequantity of vaccine to be administered will vary for the patient beingtreated and will vary from about 100 ng/kg of body weight to 100 mg/kgof body weight per day and preferably will be from 10 mg/kg to 10 mg/kgper day.

As indicated above, standard molecular biology techniques known in theart and not specifically described can be generally followed as inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York (1989), and in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989) and in Perbal, A Practical Guide to Molecular Cloning, John Wiley& Sons, New York (1988), and in Watson et al., Recombinant DNA,Scientific American Books, New York and in Birren et al. (eds) GenomeAnalysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring HarborLaboratory Press, New York (1998) and methodology as set forth in U.S.Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659; and 5,272,057, thecontents of which are incorporated herein by reference in theirentirety. Polymerase chain reaction (PCR) can be carried out generallyas in PCR Protocols: A Guide To Methods And Applications, AcademicPress, San Diego, Calif. (1990). In-situ (In-cell) PCR in combinationwith Flow Cytometry can be used for detection of cells containingspecific DNA and mRNA sequences (Testoni et al., 1996, Blood 87:3822).

As used herein, the term “gene therapy” refers to the transfer ofgenetic material (e.g., DNA or RNA) of interest into a host to treat orprevent a genetic or acquired disease or condition phenotype. Thegenetic material of interest encodes a product (e.g., a protein,polypeptide, peptide or functional RNA) whose production in vivo isdesired. For example, in addition to the nucleotide encoding 2-5 AS, ora catalytically active fragment thereof, the genetic material ofinterest can encode a hormone, receptor, or other enzyme, polypeptide orpeptide of therapeutic value. For a review see, in general, the text“Gene Therapy” (Advances in Pharmacology 40, Academic Press, 1997).

Two basic approaches to gene therapy have evolved: (1) ex vivo and (2)in vivo gene therapy. In ex vivo gene therapy, cells are removed from apatient, and while being cultured are treated in vitro. Generally, afunctional replacement gene is introduced into the cell via anappropriate gene delivery vehicle/method (transfection, transduction,homologous recombination, etc.) and an expression system as needed andthen the genetically modified cells are expanded in culture and returnedto the host/patient. These genetically reimplanted cells produce thetransfected gene product in situ. Alternatively, a xenogenic orallogeneic donor's cells can be genetically modified with the nucleotidesequence in vitro and subsequently administered to the patient.

In in vivo gene therapy, target cells are not removed from the patient;rather, the gene to be transferred is introduced into the cells of therecipient organism in situ, that is within the recipient. Alternatively,if the host gene is defective, the gene is repaired in situ. Thesegenetically modified cells produce the transfected gene product in situ.

The gene expression vehicle is capable of delivery/transfer ofheterologous nucleic acids into a host cell. As indicated previously,the expression vehicle may include elements to control targeting,expression and transcription of the nucleotide sequence in a cellselective or tissue-specific manner, as is known in the art. It shouldbe noted that often the 5′UTR and/or 3′UTR of the gene may be replacedby the 5′UTR and/or 3′UTR of the expression vehicle. Therefore as usedherein the expression vehicle may, as needed, not include the 5′UTRand/or 3′UTR and only include the specific amino acid coding region.

The expression vehicle can include a promoter for controllingtranscription of the heterologous material and can be either aconstitutive or inducible promoter to allow selective transcription.Enhancers that may be required to obtain necessary transcription levelscan optionally be included. Enhancers are generally any non-translatedDNA sequence which works contiguously with the coding sequence (in cis)to change the basal transcription level dictated by the promoter. Theexpression vehicle can also include a selection gene as described hereinbelow.

Vectors can be introduced into cells or tissues by any one of a varietyof known methods within the art. Such methods can be found generallydescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992); in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press,Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press, AnnArbor, Mich. (1995); Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Butterworths, Boston Mass. (1988); and Gilboa et al. (1986)and include, for example, stable or transient transfection, lipofection,electroporation and infection with recombinant viral vectors. Inaddition, see U.S. Pat. No. 4,866,042 for vectors involving the centralnervous system and also U.S. Pat. Nos. 5,464,764 and 5,487,992 forpositive-negative selection methods.

Introduction of nucleic acids by infection offers several advantagesover the other listed methods. Higher efficiency can be obtained due totheir infectious nature. Moreover, viruses are very specialized andtypically infect and propagate in specific cell types. Thus, theirnatural specificity can be used to target the vectors to specific celltypes in vivo or within a tissue or mixed culture of cells. Viralvectors can also be modified with specific receptors or ligands to altertarget specificity through receptor mediated events.

A specific example of a DNA viral vector for introducing and expressingrecombinant nucleotide sequences is the adenovirus derived vectorAdenop53TK. This vector expresses a herpes virus thymidine kinase (TK)gene for either positive or negative selection and an expressioncassette for desired recombinant sequences. This vector can be used toinfect cells that have an adenovirus receptor which includes mostcancers of epithelial origin as well as others. This vector as well asothers that exhibit similar desired functions can be used to treat amixed population of cells and can include, for example, an in vitro orex vivo culture of cells, a tissue or a human subject.

Additional features can be added to the vector to ensure its safetyand/or enhance its therapeutic efficacy. Such features include, forexample, markers that can be used to negatively select against cellsinfected with the recombinant virus. An example of such a negativeselection marker is the TK gene described above that confers sensitivityto the antibiotic gancyclovir. Negative selection is therefore a meansby which infection can be controlled because it provides induciblesuicide through the addition of antibiotic. Such protection ensures thatif, for example, mutations arise that produce altered forms of the viralvector or recombinant sequence, cellular transformation will not occur.Features that limit expression to particular cell types or tissue typescan also be included. Such features include, for example, promoter andregulatory elements that are specific for the desired cell type ortissue type.

In addition, recombinant viral vectors are useful for in vivo expressionof a desired nucleic acid because they offer advantages such as lateralinfection and targeting specificity. Lateral infection is inherent inthe life cycle of, for example, retrovirus and is the process by which asingle infected cell produces many progeny virions that bud off andinfect neighboring cells. The result is that a large area becomesrapidly infected, most of which was not initially infected by theoriginal viral particles. This is in contrast to vertical-type ofinfection in which the infectious agent spreads only through daughterprogeny. Viral vectors can also be produced that are unable to spreadlaterally. This characteristic can be useful if the desired purpose isto introduce a specified gene into only a localized number of targetedcells.

As described above, viruses are very specialized infectious agents thathave evolved, in many cases, to elude host defense mechanisms.Typically, viruses infect and propagate in specific cell types. Thetargeting specificity of viral vectors utilizes its natural specificityto specifically target predetermined cell types and thereby introduce arecombinant gene into the infected cell. The vector to be used in themethods of the present invention will depend on desired the cell type orcell types to be targeted and will be known to those skilled in the art.For example, if RSV infection is to be inhibited (i.e., treated orprevented), then a vector specific for such respiratory mucosalepithelial cells would preferably be used.

Retroviral vectors can be constructed to function either as infectiousparticles or to undergo only a single initial round of infection. In theformer case, the genome of the virus is modified so that it maintainsall the necessary genes, regulatory sequences and packaging signals tosynthesize new viral proteins and RNA. Once these molecules aresynthesized, the host cell packages the RNA into new viral particlesthat are capable of undergoing further rounds of infection. The vector'sgenome is also engineered to encode and express the desired recombinantnucleotide sequence. In the case of non-infectious viral vectors, thevector genome is usually mutated to destroy the viral packaging signalthat is required to encapsulate the RNA into viral particles. Withoutsuch a signal, any particles that are formed will not contain a genomeand therefore cannot proceed through subsequent rounds of infection. Thespecific type of vector will depend upon the intended application. Theactual vectors are also known and readily available within the art orcan be constructed by one skilled in the art using well-knownmethodology.

The recombinant vector can be administered in several ways. If viralvectors are used, for example, the procedure can take advantage of theirtarget specificity and consequently, do not have to be administeredlocally at the diseased site. However, local administration can providea quicker and more effective treatment, administration can also beperformed by, for example, intravenous or subcutaneous injection intothe subject. Injection of the viral vectors into a spinal fluid can alsobe used as a mode of administration, especially in the case of RNA virusinfections of the central nervous system. Following injection, the viralvectors will circulate until they recognize host cells with theappropriate target specificity for infection.

An alternate mode of administration can be by direct inoculation locallyat the site of the disease or pathological condition or by inoculationinto the vascular system supplying the site with nutrients or into thespinal fluid. Local administration is advantageous because there is nodilution effect and, therefore, a smaller dose is required to achieveexpression in a majority of the targeted cells. Additionally, localinoculation can alleviate the targeting requirement required with otherforms of administration since a vector can be used that infects allcells in the inoculated area. If expression is desired in only aspecific subset of cells within the inoculated area, then promoter andregulatory elements that are specific for the desired subset can be usedto accomplish this goal. Such non-targeting vectors can be, for example,viral vectors, viral genome, plasmids, phagemids and the like.Transfection vehicles such as liposomes and colloidal polymericparticles can also be used to introduce the non-viral vectors describedabove into recipient cells within the inoculated area. Such transfectionvehicles are known to those skilled within the art.

Direct DNA inoculations can be administered as a method of vaccination.Plasmid DNAs encoding influenza virus hemagglutinin glycoproteins havebeen tested for the ability to provide protection against lethalinfluenza challenges. In immunization trials using inoculations ofpurified DNA in saline, 67-95% of test mice and 25-63% of test chickenswere protected against the lethal challenge. Good protection wasachieved by intramuscular, intravenous and intradermal injections. Inmice, 95% protection was achieved by gene gun delivery of 250-2500 timesless DNA than the saline inoculations. Successful DNA vaccination bymultiple routes of inoculation and the high efficiency of gene-gundelivery highlight the potential of this promising new approach toimmunization. Plasmid DNAs expressing influenza virus hemagglutininglycoproteins have been tested for their ability to raise protectiveimmunity against lethal influenza challenges of the same subtype. Intrials using two inoculations of from 50 to 300 micrograms of purifiedDNA in saline, 67-95% of test mice and 25-63% of test chickens have beenprotected against a lethal influenza challenge. Parenteral routes ofinoculation that achieved good protection included intramuscular andintravenous injections. Successful mucosal routes of vaccinationincluded DNA drops administered to the nares or trachea. By far, themost efficient DNA immunizations were achieved by using a gene gun todeliver DNA-coated gold beads to the epidermis. In mice, 95% protectionwas achieved by two immunizations with beads loaded with as little as0.4 micrograms of DNA. The breadth of routes supporting successful DNAimmunizations, coupled with the very small amounts of DNA required forgene-gun immunizations, highlight the potential of this remarkablysimple technique for the development of subunit vaccines. In contrast tothe DNA based antigen vaccines, the present invention provides thedevelopment of an intranasal gene transfer method using 2-5 AS, or acatalytically active fragment thereof, which can be used as aprophylaxis against multiple respiratory infections. In a preferredembodiment, the preventative and therapeutic method is used againstrespiratory RNA viral infection, most preferably against RSV.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

What is claimed is:
 1. A method of increasing expression of 2′-5′oligoadenylate synthetase in a mammal, said method comprisingadministering to the mammal a nucleotide sequence encoding a 2′-5′oligoadenylate synthetase, or at least one enzymatically active fragmentthereof, wherein the nucleotide sequence is expressed in the mammal. 2.The method of claim 1, wherein the enzymatically active fragmentcomprises an enzymatically active subunit of 2′-5′ oligoadenylatesynthetase selected from the group consisting of p40, p69, and p100. 3.The method according to claim 1, wherein the nucleotide sequence isadministered to the mammal within a viral vector.
 4. The method of claim1, wherein the nucleotide sequence is administered to the mammalintranasally.
 5. The method of claim 1, wherein the expression of 2′-5′oligoadenylate synthetase is increased to treat infection of the mammalby an RNA virus, and wherein the RNA virus is a type that transientlyproduces double-stranded RNA during intermediate replication.
 6. Themethod of claim 5, wherein the mammal is a human.
 7. The method of claim6, wherein the human is a child.
 8. The method of claim 6, wherein thehuman is elderly.